Methods for tissue welding using laser-activated protein solders

ABSTRACT

Various tissue glues have drawback such as toxicity, causing inflammatory reactions or insufficient bonding strength. The invention is directed to methods of form tissue adhesion by administering to tissues compositions comprising proteins conjugated to one or more novel photosensitizers and irradiating the composition. The composition may further comprise one or more proteins not conjugated to the photosensitizer. Additionally, the present invention relates to compositions and methods wherein increased ratios of protein to photosensitizer enhance weld strength.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of medical gluesand adhesives. More particularly, it concerns methods and compositionsfor sealing of wounds and incisions. In certain aspects, the inventionconcerns adhesion of two or more tissue samples using proteinaceous,and/or lipoproteinaceous compositions conjugated and/or mixed with aphotosensitizer or dye upon irradiation.

2. Description of Related Art

Healing and sealing tissue wounds remains a problem in medical practice.To enhance tissue healing, much effort has gone towards producing abiocompatible slow release formulation which can be introduced into atissue defect and that will release biologically active growth factor ata steady rate for the required time. These formulations were designed topromote tissue growth or healing. Examples of these vehicles includebiodegradible gelatin hydrogel (Yamamoto et al., 2000), hyaluronan(Mohammad et al., 2000), fibrin glue (Cheng et al., 1998), fibrinderivatives (Sakiyama-Elbert & Hubbell, 2000), alginate microspheres(Nehra et al., 1999), carbopol gel (Sheardown et al., 1997), derivatizeddextrans (Tardieu et al., 1992), calcium alginate beads (Downs et al.,1992). It should be noted that many of these slow release vehicles donot actually bind to the tissue, they merely “sit” in the defect andslowly biodegrade. However, a TGF-beta 1 and indocyanine green albuminsolder have been used in incisions in pig skin (Poppas et al., 1996). Asignificant increase in wound healing strength was found using TGF-betacontaining solder compared to solder alone.

To replace or promote healing in damaged tissue, various forms of tissuetransplants have been conducted. However, cell transplantation oftenrequires that that the donor cells retain their polarity and function,avoid formation of clumps or multilayers, and maintain their viability.In certain tissues, a biodegradable matrix has been used to transplantcells. Three-dimensional cell culture systems with various attachmentsubstrates offer new probabilities for long-term viability and donorcell functions (Wintermantel et al., 1992; Fawcett et al., 1995; Spierand Maroudas, 1991; Peshwa et al., 1996; Rezai et al., 1997; Ruoslahtiand Hayman, 1992; Spector et al., 1993; Hoffman, 1994; Kleinman et al.,1998). Successful use of 3-dimensional micro-carriers fortransplantation to the liver (Wintermantel et al., 1992) and brain(Fawcett et al., 1995) has been reported by several investigators. In3-dimensional micro-carriers, the cultured cells are distributed at theouter surfaces and within the body of the particles (Spier and Maroudas,1991). In a 3-dimensional carrier, more cell contacts are generatedcompared with a monolayer state, thereby facilitating cell proliferationand spreading (Peshwa et al., 1996; Rezai et al., 1997). The chemistryof the extracellular matrix itself can also modulate various aspects ofcell behavior, including adhesion, proliferation, and migration(Ruoslahti and Hayman, 1992).

Successful retinal pigment epithelium (RPE) transplantation requirescell attachment to a substrate prevents RPE apoptosis andde-differentiation after transplantation (Tezel and Del Priore, 1997; Hoet al., 1996). Subretinal provision of RPE cells has been carried out inthe form of a cell suspension, RPE patches, or RPE cells grown onartificial substrates (Li and turner, 1991; Sheedlo et al., 1989;Gabrielian et al., 1999; Bhatt et al., 1994). Cell suspension provisionhas the limitations of reflux from the iatrogenic retinotomy site andirregular distribution of the donor cells in the subretinal space(Wongpichedchai et al., 1992). Retinal pigment epithelium patch grafts,although probably the most physiologic, have not been shown toproliferate in vivo (Gouras et al., 1994; Berglin et al., 1997).

Sealing tissue wounds usually involves sutures and other mechanicalseals. Alternative methods to the traditional mechanical means ofclosing incisions, wounds, and anastomoses have received attention.These may be divided into three groups: first, biological glues (Basu etal., 1995) such as fibrin sealant (Sierra, 1993) and gelatin-resorcinolglue (Albes et al., 1993); second, a technique known as laser tissuewelding, which relies on carbon dioxide (Rooke et al., 1993) or Nd:YAG(Back et al., 1994) lasers to produce thermal effects to attach tissuesurfaces; and third, chromophore-assisted laser welding (Bass and Treat,1995) using protein solders that contain a light-absorbing dye togetherwith a laser that emits the appropriate wavelength light. This pairingis most commonly that of fluorescein and a 532-nm frequency-doubledNd:YAG laser, or indocyanine green and an 805-nm diode laser (Wright andPoppas, 1997).

Alternative tissue adhesives have drawbacks. Cyanoacrylate glues, whichhave been most frequently used in ophthalmology (Leahey et al., 1993)can be toxic, causing inflammatory reactions and are nonbiodegradable(Siegal and Zaidman, 1989). Fibrin sealants (Spontnitz, 1995) are notparticularly effective, form bonds of insufficient strength (Basu etal., 1995; Siedentop et al., 1988), present the possibility of viralinfection if prepared from pooled human plasma, and may inhibit wouldhealing (van der Ham et al., 1993). Resorcinol gelatin sealants (Albeset al., 1993) can damage tissue because they contain formaldehyde(Ennker et al., 1994). However, laser-activated tissue solders allowssafe preparation and sterilization of the material, because it isactivated only under laser illumination and is thought unlikely to leadto tissue toxicity (Bass and Treat, 1995).

Laser tissue welding have been used in urology (Kirsch et al., 1997),vascular surgery (Ashton et al., 1991), neurosurgery (Menovsky et al.,1995), and orthopedics (Forman et al., 1995). Ophthalmologicapplications of laser welding with chromophore-assisted protein solderhave included sealing cataract incisions (Eaton et al., 1991) andscleral tunnel incisions (Kim et al., 1995) and bonding syntheticepikeratoplasty lenticules to the cornea (Gailitis et al., 1990).

Laser tissue welding without added dye must proceed through a purelythermal mechanism (Schober et al., 1986), whereby the edges of thecollagen are partially “unraveled” and can then recombine to formnoncovalent bonds (Pearce and Thomsen, 1993). It was thought thatdye-assisted welding with protein solders also proceeded through athermal mechanism, with the chromophore-absorbing energy, releasing itas heat, denaturing the protein in the solder and forming noncovalentbonds between the added protein solder and the tissue collagen (Small etal., 1997). A mixture of cryoprecipitated fibrinogen and a dye thatabsorbs laser energy and releases it in the form of heat at the woundinterface has been used in tissue adhesion (Moazami, et al., 1990; Oz etal., 1990).

However, results with the two dyes most commonly used for tissuewelding, fluorescein and indocyanine green, have produced evidence thatphotochemical processes occur as well. It has been reported thatfluoresceindextran in the rat mesentery lymphatics when illuminatedproduce changes that could be attributed to singlet oxygen (Zhang etal., 1997). Studies with indocyanine green in vitro have shown that ithas a triplet yield of 0.11, and singlet oxygen can be detected bytime-resolved luminescence techniques (Baumier et al., 1997; Fickwelleret al., 1997). Laser welding with a biologic tissue glue consisting of18% fibrinogen with 2.6 mg/ml r-5-P showed reduction of the weldstrength in the presence of azide which is evidence of singlet oxygeninvolvement in the weld formation (Khadem et al., 1994).

These chemicals that cause photo-oxidative effects when exposed tovisible light have been called “photosensitizers” (Chacon et al., 1988;Tanielian C., 1986; Foote, C. S., 1976). There are two main classes ofphotosensitizer: tetrapyrroles including porphyrins, chlorins,bacteriochlorins, phthalocyanines, naphthalocyanines, texaphyrins,verdins, purpurins; pheophorbides, etc; and non-tetrapyrrole dyes,including flavins, xanthenes, thiazines, selenium and telluriumanalogues of thiazines, azines, triarylmethanes, etc. Fluorescein is axanthene but is not considered a photosensitizer because it releasesabsorbed energy primarily in the form of heat and fluorescence. Some ofthese dyes have been evaluated with proteins as tissue glues withvarying success (U.S. Pat. No. 5,552,452).

Chlorin_(e6) (C_(e6)) has been investigated as a photosensitizer forphotodynamic therapy both as the free dye (Kostenich et al., 1994) andconjugated to proteins, (Schmidt-Erfurth et al., 1997), macromolecules(Soukos et al., 1997) and particles (Bachor et al., 1991). Covalentconjugates between C_(e6) and monoclonal antibodies (Hamblin et al.,1996) and poly-_(L)-amino acids (Soukos et al., 1997) for thephotodynamic therapy of cancer have been described. C_(e6) is usuallythought to act as a photosensitizer by transferring energy from thetriplet state to the ground state of molecular oxygen, producing theexited singlet oxygen molecule, a process known as type IIphotosensitization (Ochsner, 1997). Singlet oxygen can then react withcertain amino acids in proteins, particularly histidine, tryptophan,tyrosine, cysteine, and methionine (Dubbelman et al., 1978). Onemechanism that has been elucidated for the formation of intermolecularprotein cross-links is the reaction of oxidized histidine with freeamino groups of lysines on neighboring proteins (Verweij et al., 1981),but it is recognized that other mechanisms must operate as well. Thereis another possible photo-oxidation pathway involving electron transferfrom the photosensitizer triplet state producing either a radical cationor a radical anion, which is known as type I photosensitization (Zhangand Xu, 1994). These radical ions can then react further with oxygenproducing carbon and oxygen centered radicals and superoxide anions(Laustrait, 1986). A mechanism for the radical mediated cross-linking ofproteins involves the formation of dityrosine (Gill et al., 1997) byphenolic coupling of tyrosine residues on neighboring chains.

Despite these advances in the understanding of tissue healing, tissuetransplantation and tissue welding mechanisms, their exists a need forimproved methods to heal, transplant and/or weld tissue. Improvedmethods to promote tissue healing or aid in successful tissuetransplants would provide significant benefits in the art. Tissue weldswith improved strength would resist tearing under stress. Additionally,there exists a need for methods and compositions of tissue welding,wound healing and tissue transplantation that are easy to handle duringsurgery, and possess a reduced toxicity or scaring potential.

SUMMARY OF THE INVENTION

The present invention overcomes the deficiencies of the prior art byproviding novel compositions and methods for tissue welding. Theinvention also provides compositions and methods for administering anactive agent to a tissue. Such active agents may be living cells. Thus,in certain embodiments, the invention provides a method to transplanttissue. Such transplanted cells may be formed into a desired shape, suchas a monolayer.

The invention first provides a method to weld tissue together,comprising the steps of: applying to at least one tissue a compositioncomprising at least one photosensitizer and at least one proteinaceouscompound or at least one lipid; and irradiating the composition withelectromagnetic energy; wherein the irradiating promotes adhesion of thetissue to at least a second tissue. In certain embodiments, thephotosensitizer is a cationic azine mon-azo dye or derivative thereof.In certain aspects, the cationic azine mono-azo dye is neutral red orJanus Green. In other embodiments, the photosensitizer is atri-arylmethane dye or derivative thereof. In particular aspects, thetri-arylmethane dye is Malachite Green, Brilliant Green, Crystal Violet,basic fuschin, pararosaniline acetate, methyl green or new fuschin. Incertain aspects, the tri-arylmethane dye is a zwitterionictriarylmethane dye, such as patent blue VF. In other embodiments, thephotosensitizer is a tetrapyrrole or a derivative thereof. In certainaspects, the tetrapyrrole is a porphyrin, chlorin, bacteriochlorin,phthalocyanine, naphthalocyanine, texaphyrin, verdin, purpurin orpheophorbide. In some aspects, the chlorin is chlorin_(e)6. In otheraspects, the phthalocyanine is a Zn(II)-phthalocyanine, an aluminumsulfonated and disulfonated phthalocyanine or a phthalocyanine without ametal substituent. In certain aspects, the naphthalocyanine is asulfonated aluminum naphthalocyanine. In other aspects, the pheophorbideis a pyropheophorbide. In certain embodiments, the photosensitizer is acationic thiazine dye or derivative thereof. In particular embodiments,the cationic thiazine dye is Azure A, Azure B, Azure C, Brilliant Green,Crystal Violet or Patent Blue VF.

In certain embodiments, the composition further comprises at least asecond photosensitizer. In particular aspects, the at least a secondphotosensitizer is a cationic azine mon-azo dye, a tri-arylmethane dye,a tetrapyrrole, a cationic thiazine dye, xanthine, an anthracenedione,an anthrapyrazole, an aminoanthraquinone, a phenoxazine dye, aphenothiazine derivative, a chalcogenapyrylium dye or derivativesthereof.

In certain embodiments, the composition comprises at least oneproteinaceous compound. In certain aspects, the proteinaceous compoundcomprises at least one peptide, polypeptide or protein. In particularaspects, the protein is albumin, fibrinogen or gelatin.

In some embodiments, the composition is a non-covalent mixture. In otherembodiments, at least one covalent bond conjugates the photosensitizerto the proteinaceous material or the lipid. In particular aspects, thecovalent bond is part of a linking moeity.

In other embodiments, the composition comprises at least a secondproteinaceous compound not covalently conjugated to the photosensitizer.In certain aspects, the proteinaceous compound covalently conjugated tothe photosensitizer is the same type as the proteinaceous compound notcovalently conjugated to the photosensitizer, such as albumin for bothproteinaceous compounds.

In certain embodiments, the ratio of total proteinaceous molecules inthe composition and the at least one photosensitizer is from about 100:1to about 1:100. In certain aspects, the ratio of total proteinaceousmolecules in the composition and the at least one photosensitizer isfrom about 10:1 to about 1:10. In other aspects, the ratio of totalproteinaceous molecules in the composition and the at least onephotosensitizer is from about 3:1 to about 1:1. In a particular aspect,the ratio of total proteinaceous molecules in the composition and the atleast one photosensitizer is about 2:1.

In some embodiments, the composition comprises at least one lipid. Incertain aspects, the lipid further comprises at least one proteinaceouscompound. In other aspects, the proteinaceous compound is a lipoprotein.

In certain embodiments, the composition further comprises at least onetherapeutic agent. In particular aspects, the agent is a chemical, adrug, a proteinaceous molecule, a nucleic acid, a lipid, an antibody, anantigen, a hormone, a nutritional substance, a cell or a combinationthereof In certain aspects, the hormone is a growth factor, includingbut not limited to transforming growth factor beta, basic fibroblastgrowth factor, epidermal growth factor, vascular endothelial growthfactor, nerve growth factor, acidic fibroblast growth factor, insulinlike growth factor, heparin binding growth factor, brain-derivedneurotrophic factor, glial cell line-derived neurotrophic factor,platelet-derived growth factor, leukemia inhibitory factor orcombination thereof. In other aspects, the agent is a cell, includingbut not limited to an embryonic cell.

In other embodiments, the tissue is skin, bone, neuron, axon, cartilage,blood vessel or cornea. In certain aspects, the second tissue is thesame tissue type as the at least a first tissue, while in other aspects,the second tissue is a different tissue type as the at least one tissue.

In certain embodiments, the composition is applied at of from about 10mg the composition per cm² of the tissue to about 500 mg the compositionper cm² of the tissue. In particular aspects, the composition is appliedof from about 20 mg the composition per cm² of the tissue to about 100mg the composition per cm² of the tissue.

In particular embodiments, the composition has a viscosity of about 40to about 100 poise before the irradiation.

The invention next provides a method to weld tissue together, comprisingthe steps of: applying to at least one tissue a composition comprisingat least one photosensitizer; and irradiating the composition withelectromagnetic energy; wherein the photosensitizer is a cationic azinemon-azo dye, a tri-arylmethane dye, a chlorine, a tetrapyrrole, acationic thiazine dye, or derivatives thereof; and wherein theirradiating promotes adhesion of the tissue to at least a second tissue.In certain embodiments, the photosensitizer is neutral red, Janus Green,Malachite Green, Brilliant Green, Crystal Violet, basic fuschin,pararosaniline acetate, methyl green, new fuschin, patent blue VF12,chlorin_(e)6, Azure A, Azure B, Azure C, Brilliant Green, Crystal Violetor Patent Blue VF. In particular aspects, the photosensitizer is JanusGreen, Malachite Green or chlorin_(e)6.

In certain embodiments, the composition further comprises at least asecond photosensitizer. In particular aspects, the at least a secondphotosensitizer is a cationic azine mon-azo dye, a tri-arylmethane dye,a tetrapyrrole, a cationic thiazine dye, xanthine, an anthracenedione,an anthrapyrazole, an aminoanthraquinone, a phenoxazine dye, aphenothiazine derivative, a chalcogenapyrylium dye or derivativesthereof.

In other embodiments, the composition comprises at least oneproteinaceous compound or lipid.

In certain embodiments, the method of claim 56, wherein the compositionis a non-covalent mixture. In some embodiments, the at least onecovalent bond conjugates the photosensitizer to the proteinaceousmaterial or the lipid. In particular aspects, the covalent bond is partof a linking moeity.

In some embodiments, the composition comprises at least a secondproteinaceous compound not covalently conjugated to the photosensitizer.In certain aspects, the proteinaceous-compound covalently conjugated tothe photosensitizer is the same type as the proteinaceous compound notcovalently conjugated to the photosensitizer.

In particular embodiments, the composition further comprises at leastone therapeutic agent. In some aspects, the agent is a chemical, a drug,a proteinaceous molecule, a nucleic acid, a lipid, an antibody, anantigen, a hormone, a nutritional substance, a cell or a combinationthereof.

The invention further provides a method to deliver a therapeutic agentto at least one living cell, comprising the steps of: applying to atleast one cell a composition comprising at least one at least onephotosensitizer and at least a therapeutic agent; and irradiating thecomposition with electromagnetic energy; wherein the irradiatingpromotes adhesion of the composition to the cell, and wherein the agentis thereby contacted with the cell. In certain aspects, thephotosensitizer is a cationic azine mon-azo dye, a tri-arylmethane dye,a chlorine, a tetrapyrrole, a cationic thiazine dye, or derivativesthereof. In other aspects, the photosensitizer is neutral red, JanusGreen, Malachite Green, Brilliant Green, Crystal Violet, basic fuschin,pararosaniline acetate, methyl green, new fuschin, patent blue VF12,chlorin_(e)6, Azure A, Azure B, Azure C, Brilliant Green, Crystal Violetor Patent Blue VF. In particular aspects, the photosensitizer is JanusGreen, Malachite Green or chlorin_(e)6.

In certain embodiments, the composition further comprises at least asecond photosensitizer. In some aspects, the at least a secondphotosensitizer is a cationic azine mon-azo dye, a tri-arylmethane dye,a tetrapyrrole, a cationic thiazine dye, xanthine, an anthracenedione,an anthrapyrazole, an aminoanthraquinone, a phenoxazine dye, aphenothiazine derivative, a chalcogenapyrylium dye or derivativesthereof.

In particular embodiments, the composition comprises at least oneproteinaceous compound or lipid.

In other embodiments, the composition is a non-covalent mixture. In someembodiments, at least one covalent bond conjugates the photosensitizerto the proteinaceous material or the lipid. In particular aspects, thecovalent bond is part of a linking moeity. In certain aspects, thecomposition comprises at least a second proteinaceous compound notcovalently conjugated to the photosensitizer. In other aspects, theproteinaceous compound covalently conjugated to the photosensitizer isthe same type as the proteinaceous compound not covalently conjugated tothe photosensitizer.

In some embodiments, the agent is a chemical, a drug, a proteinaceousmolecule, a nucleic acid, a lipid, an antibody, an antigen, a hormone, anutritional substance, a cell or a combination thereof. In particularaspects, the hormone is a growth factor. In other aspects, the growthfactor is transforming growth facto beta, basic fibroblast growthfactor, epidermal growth factor, vascular endothelial growth factor,nerve growth factor, acidic fibroblast growth factor, insulin likegrowth factor, heparin binding growth factor, brain-derived neurotrophicfactor, glial cell line-derived neurotrophic factor, platelet-derivedgrowth factor, leukemia inhibitory factor or combination thereof. Inother aspects, the agent is a cell. In specific aspects, the cell is anembryonic cell.

In certain embodiments, a tissue may comprise, but is not limited to,skin, bone, neuron, axon, cartilage, blood vessel, cornea, muscle,facia, brain, prostate, breast, endometrium, lung, pancreas, smallintestine, blood, liver, testes, ovaries, cervix, colon, skin, stomach,esophagus, spleen, lymph node, bone marrow, kidney, peripheral blood,embryonic or ascite tissue, and all cancers thereof. In certainembodiments, a cell may comprise, but is not limited to, skin, bone,neuron, axon, cartilage, blood vessel, cornea, muscle, facia, brain,prostate, breast, endometrium, lung, pancreas, small intestine, blood,liver, testes, ovaries, cervix, colon, skin, stomach, esophagus, spleen,lymph node, bone marrow, kidney, peripheral blood, embryonic or ascitecell, and all cancers thereof.

The invention additionally provides a tissue glue/biomatrix composition,comprising at least one photosensitizer and at least one proteinaceouscompound or at least one lipid. In certain embodiments, thephotosensitizer is a cationic azine mon-azo dye, a tri-arylmethane dye,a chlorine, a tetrapyrrole, a cationic thiazine dye, or derivativesthereof. In other embodiments, the photosensitizer is neutral red, JanusGreen, Malachite Green, Brilliant Green, Crystal Violet, basic fuschin,pararosaniline acetate, methyl green, new fuschin, patent blue VF12,chlorin_(e)6, Azure A, Azure B, Azure C, Brilliant Green, Crystal Violetor Patent Blue VF. In particular aspects, the photosensitizer is JanusGreen, Malachite Green or chlorin_(e)6. In other embodiments, thecomposition further comprises a therapeutic agent. In certain aspects,the agent is a chemical, a drug, a proteinaceous molecule, a nucleicacid, a lipid, an antibody, an antigen, a hormone, a nutritionalsubstance, a cell or a combination thereof.

The invention next provides a tissue glue/biomatrix composition,comprising at least one photosensitizer, wherein the photosensitizer isa cationic azine mon-azo dye, a tri-arylmethane dye, a chlorine, atetrapyrrole, a cationic thiazine dye, or derivatives thereof. Incertain embodiments, the composition further comprises at least onetherapeutic agent, a proteinaceous compound or lipid.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

Following long-standing patent law convention, the word “a” and “an”mean “one or more” in this specification, including the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Reaction scheme used to prepare covalent protein C_(e6)conjugates. DCC, dicyclohexylcarbodiimide; NHS, N-hydroxysuccinimide,DMSO, dimethyl sulfoxide.

FIG. 2. Absorption spectra of three solder preparations. Solders werediluted in 0.1 M NaOH-1% sodium dodecyl sulfate between 500 and 1200times.

FIG. 3. Fluence dose-response curve showing weld leaking strength.Incisions were closed by using the specified solder preparations and anarray of applied total energies. Each point is the mean of leakingpressures from two or three eyes. Bars, ±SEM.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Tissue welding with the aid of laser or light-activated solders wouldallow sutureless surgery, as well as repair of certain wounds that aredifficult or impossible to close by standard suture techniques. This isuseful in the field of ophthalmology, because sutures, staples, andclips all involve additional tissue injury, and a foreign body responsethat can lead to increased inflammation, scarring and stenosis. Inaddition, gluing or welding tissue may reduce wound slippage and renderthe wound impermeable to microorganisms. The ideal material to use as aglue or solder should be strong, effective, nontoxic, biodegradable andavailable in a sterile preparation. For the solder preparation to beeffective, it should also have a sufficiently high viscosity to enableit to stay in contact with the wound during welding. The composition maybe applied by any method described herein or would be know to one ofordinary skill in the art, including but not limited to delivery througha convenient devise such as a needle. The ideal consistency wouldtherefore be thixotropic (i.e., a material that has lower viscosity athigher shear stresses). There are many potential applications of thistechnology in ophthalmology including the repair of leaking filteringblebs (Zalta and Wieder, 1991), corneal ulcers (Golubovic and Parunovic,1991), and scleromalacia perforans (Enzenauer et al., 1992). It may beused in construction of a temporary tarsorrhaphy (Donnenfeld et al.,1991), and the reinforcement of sclera in patients with thin sclera(Sternberg et al., 1988) or staphyloma (Coroneo et al., 1988).

To provide improved methods and compositions for laser mediated tissuewelding, various compositions of dyes and proteins conjugated togetherwere examined. Tissue glue covalent bond formation mediated by aphotodynamic process were compared in a protein solder comprisingprotein and C_(e6) covalent conjugates to noncovalent mixturescomprising protein and C_(e6). The inventors reasoned that aphotosensitizer molecule already joined to the protein would be morelikely to form a bond between that protein and a neighboring proteinmolecule than a photosensitizer that had to be close to two proteinmolecules at the same time. The finding that the strength of the weldformed by the bovine serum albumin-Chlorin_(e6) (BSA-C_(e6)) conjugatewas significantly stronger than that formed by the noncovalent mixturewas demonstrated. Additionally, it was also demonstrated that aphotosensitizer widely thought to proceed through a type IIphotosensitization mechanism could form satisfactory tissue welds whenapplied in protein solder.

In particular, a photosensitizer which has known triplet and singletoxygen quantum yields, namely C_(e6), was examined for its tissue weldenhancing properties. A surprising and drastic improvement in weldstrength when the protein-to-C_(e6) ratio is increased from about 100molecules of protein to about 1 molecule dye to about 1 molecule proteinto about 100 molecules dye was observed. This enhancement in weldstrength may be due to an increased likelihood of intermolecularcross-links forming between one conjugate molecule and one unconjugatedalbumin molecule than between two conjugate molecules. It iscontemplated that ranges of proteinaceous molecule and/or lipid moleculeto dye ratios that may be useful include, but are not limited to, about100:1 about 95:1, about 90:1, about 85:1, about 80:1, about 75:1, about70:1, about 65:1, about 60:1, about 55:1, about 50:1, about 45:1, about40:1, about 35:1, about 30:1, about 25:1, about 20:1, about 17:1, about15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1,about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about1:12, about 1:13, about 1:14, about 1:15, about 1:17, about 1:20, about1:25, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about1:55, about 1:60, about 1:65, about 1:70, about 1:75, about 1:80, about1:85, about 1:90, about 1:95, about 1:100, and all ranges derivabletherein. It is contemplated that ranges of proteinaceous molecule and/orlipid molecule to dye ratios of about 3:1, about 2:1, and about 1:1 arepreferable. A particularly preferred range is about 2 molecules oflipid, protein, polypeptides and/or peptides to 1 molecule of dye. Insome aspects, the proteinaceous material and/or lipid and dye are notconnected by a covalent bond, anid the composition comprises essentiallynon-covalently associated lipid, protein, polypeptide and/or peptide-dyecompositions. In other aspects, some or all of the proteinaceous and/orlipid material is covalently attached to the dye. It is contemplatedthat about 1, about 2, about 3, about 4, about 5, about 6, about 7,about 8, about 9, about 10, about 11, about 12, about 13, about 14,about 15, about 16, about 17, about 18, about 19, about 20, about 21,about 22, about 23, about 24, about 25, about 26, about 27, about 28,about 29, about 30, about 31, about 32, about 33, about 34, about 35,about 36, about 37, about 38, about 39, about 40, about 41, about 42,about 43, about 44, about 45, about 46, about 47, about 48, about 49,about 50, about 51, about 52, about 53, about 54, about 55, about 56,about 57, about 58, about 59, about 60, about 61, about 62, about 63,about 64, about 65, about 66, about 67, about 68, about 69, about 70,about 71, about 72, about 73, about 74, about 75, about 76, about 77,about 78, about 79, about 80, about 81, about 82, about 83, about 84,about 85, about 86, about 87, about 88, about 89, about 90, about 91,about 92, about 93, about 94, about 95, about 96, about 97, about 98,about 99, about 100 percent, and any range derivable therein, of the atleast one lipid, protein, polypeptide and/or peptide is covalentlyconjugated to the at least one dye before photoactivation. In otheraspects, it is contemplated that about 1, about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 11, about12, about 13, about 14, about 15, about 16, about 17, about 18, about19, about 20, about 21, about 22, about 23, about 24, about 25, about26, about 27, about 28, about 29, about 30, about 31, about 32, about33, about 34, about 35, about 36, about 37, about 38, about 39, about40, about 41, about 42, about 43, about 44, about 45, about 46, about47, about 48, about 49, about 50, about 51, about 52, about 53, about54, about 55, about 56, about 57, about 58, about 59, about 60, about61, about 62, about 63, about 64, about 65, about 66, about 67, about68, about 69, about 70, about 71, about 72, about 73, about 74, about75, about 76, about 77, about 78, about 79, about 80, about 81, about82, about 83, about 84, about 85, about 86, about 87, about 88, about89, about 90, about 91, about 92, about 93, about 94, about 95, about96, about 97, about 98, about 99, about 100 percent, and any rangederivable therein, of the at least one dye is covalently conjugated tothe at least one lipid, protein, polypeptide and/or peptide beforephotoactivation. As used herein, “any range derivable therein” meansranges selected from the numbers described in the specification. In anon-limiting example, ranges of about 76 to about 78 percent, about 33to about 98 percent, etc., of at least one dye may be covalentlyconjugated to a polypeptide in the composition before photoactivation,based on the numbers described above.

With this discovery of compositions and methods that produce greatertissue weld strength, it is now possible to provide superior tissuewelds for various medical disorders. In ophthalmology, it iscontemplated that the tissue welding compositions described herein maybe applied to help close incision or seal leaks in tissues including butnot limited to the cornea, lens, retina or ciliary body. In urology, itis contemplated the compositions and methods herein may be used to helpclose incisions, seal leaks or anastomoses in tissues including but notlimited to a kidney capsule, urethra, ureter urinary bladder. It is alsocontemplated that the compositions and methods may be used in closingreproductive tissues, such as in a vasectomy. In the gastrointestinalsystem, it is contemplated that the methods and compositions herein maybe used to help seal anastromoses in tissues including but not limitedto the esophagus, small intestine or colon, as well as help sealincisions in the pericardium, and embolize unwanted vessels. It isparticularly contemplated that the compositions and methods of thepresent invention will have applicability in endoscopic surgery. Inneurology, it is contemplated that the present invention may be used insealing leaks or incisions in dura, sealing leaks of cerebro-spinalfluid after surgery or sealing leaks of cerebral veins. In oralsettings, it is contemplated that the present invention will have use inhelping seal incisions in gingivae and pharyngotomies. In therespiratory system, it is contemplated that the compositions and methodsdescribed herein will have use in aiding the sealing of leaks in lungsafter pneumonectomies, and sealing leaks or incisions in pleura. Indermatology, it is contemplated that the present invention will helpseal incisions where sutures are contra-indicated, such as in cosmeticapplications or where hypertrophic scarring is possible. Of course, oneof skill in the art will recognize that there are additionalapplications beyond those listed above for the tissue glue or weldingmethods and compositions described herein, and all such uses areencompassed by the present invention.

A. Photosensitizers

The term “photosensitizer”, as used herein, refers to a compound capableof undergoing photoactivation as described above. Accordingly,photosensitizers can be characterized functionally as those chemicalswhich absorb electromagnetic energy, such as optical energy, and convertit primarily to chemical energy. Preferred photosensitizers for use inaccordance herewith will be compounds capable of causing aphoto-oxidative effect, and in particular, those capable of producingsinglet oxygen when exposed to light.

Photosensitizers have typically been use as cytotoxic or histotoxicagents in the presence of light. The highly reactive free radical andradical anion species produced by these agents have been shown to causeoxidative damage to human lens enzymes, (Jedziniak, 1987) and to ocularproteins of other species. This histotoxic effect has also beenexploited in their use in photodynamic cancer therapies.

There are two major types of sensitized photo-oxidative process, Type Iand Type II. The sensitizer in its ground state S₀ first absorbs lightenergy to form S₁ and T₁ which are sensitizer molecules in their excitedsinglet and triplet states, respectively. Both Type I and Type IIreactions then proceed via the triplet state because it has a muchlonger lifetime than the singlet state.

In Type I reactions, the sensitizer triplet T₁ then directly binds tothe substrate to produce substrate free radicals or radical anions. Thesubstrate radicals then can undergo further reactions, including thatwith molecular oxygen to form the superoxide anion O₂. The superoxideanion then can react in numerous ways. For example, the superoxide anioncan further react to generate hydrogen peroxide (H₂O₂) and the hydroxylradical (OH).

In Type II reactions, the sensitizer triplet most commonly reacts firstwith molecular oxygen to produce singlet oxygen (O₂). The singlet oxygenthen oxidizes the substrate to form photo-oxidation products. Directelectron transfer from triplet to oxygen also occurs to yield superoxideanions but much less efficiently.

Photosensitizers then cause oxidative damage to susceptible amino acidresidues, namely histidine, tryptophan, tyrosine, cysteine, andmethionine. They are known to cause non-disulfide covalent cross-linksin susceptible proteins (Goosey et al., 1980; Girotti et al., 1979).This process is oxygen dependent and seems to be mediated by singletoxygen rather than by superoxide anions, hydrogen peroxide, or hydroxylradicals. Natural collagen is devoid of disulfide bridges (Stimler etal., 1977).

The photosensitizer element of the composition will be used in an amounteffective to promote the formation of an adhesive upon photoactivation;i.e., to generate a photo-oxidative effect sufficient to form anadhesive. These terms are used to refer to the process by which thephotosensitizer, when exposed to light, produces singlet oxygen insufficient quantities to cause oxidative damage to amino acids. As usedherein, the term “photoactivation” is used generally to describe theprocess by which energy in the form of electromagnetic radiation isabsorbed by a compound which becomes “excited” and then functions toconvert the energy to another form of energy, preferably chemicalenergy. The chemical energy will be in the form of reactive oxygenspecies like singlet oxygen, superoxide anion, hydroxyl radical, theexcited state of the photosensitizer, photosensitizer free radical orsubstrate free radical species. The electromagnetic radiation willinclude “optical energy”, i.e., will have a wavelength in the visiblerange or portion of the electromagnetic spectrum, and will also includethe ultra violet and infra red regions of the spectrum. Thephotoactivation processes associated with the present invention may bethose which involve reduced, negligible, or no conversion or transfer ofthe absorbed energy into heat energy and, hence, are associated withincreased or enhanced transfer of the absorbed energy into chemicalenergy. The photoactivation occurs with no more than a 1-2 degreeCelsius rise in temperature, preferably no more than 1° C. rise and morepreferably no more than 0.5° C.

The damage, i.e., chemical modification, to the amino acids results inthe formation of covalent bonds or cross-links between distinct aminoacids, thus allowing the formation of a proteinaceous adhesive, seal orframework. It is the generation of this proteinaceous adhesive, usingexogenous protein, polypeptide or peptide containing compositions and/orendogenous tissue components, which allows tissues to be sealed andwounds or other incisions to be closed.

This oxidative damage takes the form of the excited state ofphotosensitizer molecules and reactive oxygen species as well assubstrate free radicals which are capable of reacting with a widevariety of compounds. The photosensitizers described herein may thusalso be characterized as compounds capable of photo-oxidatively damagingor modifying the amino acids of the protein and thus causing thepresence of highly reactive species and thus promoting the cross-linkingreactions.

Examples of photosensitive compounds for use herewith include variouslight-sensitive dyes and biological molecules such as, for example,cationic azine mon-azo dyes, including but not limited to Janus Green Bor neutral red; tri-arylmethane dyes, including but not limited toMalachite Green, Brilliant Green, Crystal Violet, basic fuschin,pararosaniline acetate, methyl green or new fuschin; zwitterionictriarylmethane dyes including but not limited to patent blue VF;chlorines or tetrapyrroles, including but not limited to chlorin_(e6);cationic thiazine dyes, including but not limited to Azure A, Azure B,Azure C, methylene blue or toluidine blue O; or any photosensitivederivatives thereof. As mentioned above, compounds which absorb andconvert electromagnetic energy, but which release a substantial amountof heat energy and do not significantly produce reactive oxygen species,are not contemplated as preferable for use in the present invention. Forexample, fluorescein is a xanthene, but is not considered aphotosensitizer as it releases absorbed energy primarily in the form ofheat and fluorescence.

Photosensitizers include, but are not limited to, hematoporphyrins, suchas hematoporphyrin HCI and hematoporphyrin esters (Dobson, J. and M.Wilson, 1992); dihematophorphyrin ester (Wilson, M. et al., 1993);hematoporphyrin IX (Russell et al., 1991, available from PorphyrinProducts, Logan, Utah) and its derivatives;3,1-mesotetrakis(o-propionamidophenyl)porphyrin; hydroporphyrins such aschlorin, herein, and bacteriochlorin of the tetra(hydroxyphenyl)porphyrin series, and synthetic diporphyrins anddichlorins; o-substituted tetraphenyl porphyrins (picket fenceporphyrins); chlorin_(e)6 monoethylendiamine monamide (CMA Goff, B. A.et al., 1994, available from Porphyrin Products, Logan, Utah);mono-1-aspartyl derivative of chlorin_(e)6, and mono- and di-1-aspartylderivatives of chlorin_(e)6; the hematoporphyrin mixture, Photofrin II(QuadraLogic Technologies, Inc., Vancouver, BC, Canada); benzoporphyrinderivatives, including benzoporphyrin derivative mono acid Ring A(BPD-MA), tetracyanoethylene adducts, dimethyl acetylene dicarboxylateadducts, Diels-Alder adducts, and monoacid ring “a” derivatives; anaphthalocyanine (Biolo, R., 1994); a Zn(II)-phthalocyanine (Shopora, M.et al., 1995); toluidine blue O (Wilson, M. et al., 1993); aluminumsulfonated and disulfonated phthalocyanine ibid.; and phthalocyanineswithout metal substituents, and with varying other substituents; a tetrasulfonated derivative; sulfonated aluminum naphthalocyanines; methyleneblue (ibid); nile blue; crystal violet; azure β chloride; and rosebengal (Wilson, M., 1994, Intl. Dent. J. 44: 187-189). Numerousphotosensitizer entities are disclosed in Wilson, M. et al., 1992, andin Okamoto, H. et al., 1992, each incorporated herein by reference.Other potential photosensitizer compositions include but are not limitedto, pheophorbides such as pyropheophorbide compounds, anthracenediones;anthrapyrazoles; aminoanthraquinone; phenoxazine dyes; phenothiazinederivatives; chalcogenapyrylium dyes including cationic selena- andtellura-pyrylium derivatives; verdins; purpurins including tin and zincderivatives of octaethylpurpurin and etiopurpurin;benzonaphthoporphyrazines; cationic imminium salts; and tetracyclines.Particularly preferred photosensitizers are Chlorin_(e)6, Janus Green,Malachite green or Phthalocyanines. It is contemplated that thecompositions disclosed herein may further comprise any combination ofphotosensitizers disclosed herein or know to one of skill in the art,and may be used in the methods of the present invention.

The choice of one or more photosensitizers will generally be made inconjunction with the choice of electromagnetic radiation contemplatedfor use in exciting the compound, as will be understood by those ofskill in the art in light of the present disclosure.

In preferred embodiments, it is contemplated that one would wish toemploy a substantially water-soluble photosensitizer, particularly wherethe photosensitizer, or combined composition, is intended for use inconnection with a substantially aqueous tissue environment such as,e.g., the eye. However, water-solubility is only required to the extentthat the photosensitizer is able to form a substantially solublecomposition on contact with either the lipid, protein, polypeptideand/or peptide containing composition, the tissue itself, or acombination of the two. In certain embodiments, the photosensitizer maybe lipophilic, and its solubility is enhanced by the presence ofamphipathic proteinaceous and/or lipid materials or constructs, or othermaterials that may aid solubility (i.e., emulsify the photosensitizer).“Substantially soluble” indicates that the various components of thecomposition and tissue are able to functionally interact and that thereis no significant particulate matter formed which may cause orcontribute to an adverse biological reaction.

B. Proteinaceous Compositions

In certain embodiments, the present invention concerns novelcompositions comprising a proteinaceous composition conjugated with atleast one photosensitizer. As used herein, a “proteinaceouscomposition”, “proteinaceous compound” or “proteinaceous material”refers to a protein of greater than about 200 amino acids or the fulllength endogenous sequence translated from a gene, a polypeptide ofgreater than about 100 amino acids, and/or a peptide of from about 3 toabout 100 amino acids. In certain embodiments the size of the at leastone protein, polypeptide or peptide chain may be, but is not limited to,about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22,about 23, about 24, about 25, about 26, about 27, about 28, about 29,about 30, about 31, about 32, about 33, about 34, about 35, about 36,about 37, about 38, about 39, about 40, about 41, about 42, about 43,about 44, about 45, about 46, about 47, about 48, about 49, about 50,about 51, about 52, about 53, about 54, about 55, about 56, about 57,about 58, about 59, about 60, about 61, about 62, about 63, about 64,about 65, about 66, about 67, about 68, about 69, about 70, about 71,about 72, about 73, about 74, about 75, about 76, about 77, about 78,about 79, about 80, about 81, about 82, about 83, about 84, about 85,about 86, about 87, about 88, about 89, about 90, about 91, about 92,about 93, about 94, about 95, about 96, about 97, about 98, about 99,about 100, about 110, about 120, about 130, about 140, about 150, about160, about 170, about 180, about 190, about 200, about 210, about 220,about 230, about 240, about 250, about 275, about 300, about 325, about350, about 375, about 400, about 425, about 450, about 475, about 500,about 525, about 550, about 575, about 600, about 625, about 650, about675, about 700, about 725, about 750, about 775, about 800, about 825,about 850, about 875, about 900, about 925, about 950, about 975, about1000, about 1100, about 1200, about 1300, about 1400, about 1500, about1750, about 2000, about 2250, about 2500 or greater residues in length,and any range derivable therein.

The term “proteinaceous composition” encompasses sequences comprising atleast one of the 20 common amino acids in naturally synthesizedproteins, or at least one modified or unusual amino acid, including butnot limited to those shown on Table 1 below.

TABLE 1 Modified and Unusual Amino Acids Abbr. Amino Acid Abbr. AminoAcid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine Baad 3-Aminoadipicacid Hyl Hydroxylysine Bala β-alanine, β-Amino-propionic acid AHylallo-Hydroxylysine Abu 2-Aminobutyric acid 3Hyp 3-Hydroxyproline 4Abu4-Aminobutyric acid, piperidinic 4Hyp 4-Hydroxyproline acid Acp6-Aminocaproic acid Ide Isodesmosine Ahe 2-Aminoheptanoic acid AIleallo-Isoleucine Aib 2-Aminoisobutyric acid MeGly N-Methylglycine,sarcosine Baib 3-Aminoisobutyric acid MeIle N-Methylisoleucine Apm2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric acidMeVal N-Methylvaline Des Desmosine Nva Norvaline Dpm 2,2′-Diaminopimelicacid Nle Norleucine Dpr 2,3-Diaminopropionic acid Orn Ornithine EtGlyN-Ethylglycine

In certain embodiments the proteinaceous composition comprises at leastone protein, polypeptide or peptide. In further embodiments theproteinaceous composition comprises a biocompatible protein, polypeptideor peptide. As used herein, the term “biocompatible” refers to asubstance which produces no significant untoward effects when appliedto, or administered to, a given animal or human subject according to themethods and amounts described herein. Such untoward or undesirableeffects are those such as significant toxicity or adverse immunologicalreactions. In preferred embodiments, biocompatible protein, polypeptideor peptide containing compositions will generally be mammalian proteinsor peptides or synthetic proteins or peptides each essentially free fromtoxins, pathogens and harmful immunogens.

Proteinaceous compositions may be made by any technique known to thoseof skill in the art, including the expression of proteins, polypeptidesor peptides through standard molecular biological techniques, theisolation of proteinaceous compounds from natural sources, or thechemical synthesis of proteinaceous materials. The nucleotide andprotein, polypeptide and peptide sequences for various genes have beenpreviously disclosed, and may be found at computerized databases knownto those of ordinary skill in the art. One such database is the NationalCenter for Biotechnology Information's Genbank and GenPept databases(http://www.ncbi.nlm.nih.gov/). The coding regions for these known genesmay be amplified and/or expressed using the techniques disclosed hereinor as would be know to those of ordinary skill in the art.Alternatively, various commercial preparations of proteins, polypeptidesand peptides are known to those of skill in the art.

In certain embodiments a proteinaceous compound may be purified.Generally, “purified” will refer to a specific or protein, polypeptide,or peptide composition that has been subjected to fractionation toremove various other proteins, polypeptides, or peptides, and whichcomposition substantially retains its activity, as may be assessed, forexample, by the protein assays, as would be known to one of ordinaryskill in the art for the specific or desired protein, polypeptide orpeptide.

In certain embodiments, the proteinaceous composition may comprise atleast one antibody. It is contemplated that antibodies to specifictissues may bind the tissue(s) and foster tighter adhesion of the glueto the tissues after welding. As used herein, the term “antibody” isintended to refer broadly to any immunologic binding agent such as IgG,IgM, IgA, IgD and IgE. Generally, IgG and/or IgM are preferred becausethey are the most common antibodies in the physiological situation andbecause they are most easily made in a laboratory setting.

The term “antibody” is used to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)₂, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like. The techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; incorporated herein by reference).

It is contemplated that virtually any protein, polypeptide or peptidecontaining component may be used in the compositions and methodsdisclosed herein. However, it is preferred that the proteinaceousmaterial is biocompatible. In certain embodiments, it is envisioned thatthe formation of a more viscous composition will be advantageous in thatwill allow the composition to be more precisely or easily applied to thetissue and to be maintained in contact with the tissue throughout theprocedure. In such cases, the use of a peptide composition, or morepreferably, a polypeptide or protein composition, is contemplated.Ranges of viscosity include, but are not limited to, about 40 to about100 poise. In certain aspects, a viscosity of about 80 to about 100poise is preferred.

Proteins and peptides suitable for use in this invention may beautologous proteins or peptides, although the invention is clearly notlimited to the use of such autologous proteins. As used herein, the term“autologous protein, polypeptide or peptide” refers to a protein,polypeptide or peptide which is derived or obtained from an organism.Organisms that may be used include, but are not limited to, a bovine, areptilian, an amphibian, a piscine, a rodent, an avian, a canine, afeline, a fungal, a plant, or a prokaryotic organism, with a selectedanimal or human subject being preferred. The “autologous protein,polypeptide or peptide” may then be used as a component of a compositionintended for application to the selected animal or human subject. Incertain aspects, the autologous proteins or peptides are prepared, forexample from whole plasma of the selected donor. The plasma is placed intubes and placed in a freezer at about −80° C. for at least about 12hours and then centrifuged at about 12,000 times g for about 15 minutesto obtain the precipitate. The precipitate, such as fibrinogen may bestored for up to about one year (Oz, 1990).

In that the compositions of the present invention are particularly.suitable for use in tissue adhesion and wound healing, preferredproteins are contemplated. Preferred protein include albumin, fibrinogenor gelatin, with albumin being most preferred.

To select other proteins, polypeptides, peptides and the like for use inthe methods and compositions of the present invention, one wouldpreferably select a proteinacous material that possesses one or more ofthe following characteristics: it forms a solution with a highpercentage of protenaceous material solubilized; it possesses a highviscosity (i.e. about 40 to about 100 poise); it has the correctmolecular charge to bind the dye if it is a non-covalent mixture (i.e.anionic protein and cationic dye, or cationic protein and anionic dye);it has the correct amino-acids present to form covalent cross-links(i.e. one or more tyrosines, histidines, tryptophans and/ormethionines); and/or it is biocompatible (i.e. from mammalian origin formammals, preferably from human origin for humans, from canine origin forcanines, etc.; it is autologous; it is non-allergenic, and/or it isnon-immunogenic).

C. Lipids

In certain embodiments, the present invention concerns novelcompositions comprising at least one lipid associated with at least onephotosensitizer. It is contemplated that lipids can chemically reactwith photosensitizers, particularly at double bonds. Thus, it iscontemplated that a photosensitizer and the at least one lipid may forma tissue glue or biomatrix upon photoactivation. Additional one or morelipoproteins embedded or associated with lipids may also react with thephotosentizer as described above for proteinaceous compositions ingeneral to form or promote the formation of a tissue adhesive orbiomatrix.

A photosensitizer associated with a lipid may be encapsulated in theaqueous interior of a liposome, interspersed within the lipid bilayer ofa liposome, attached to a liposome via a linking molecule that isassociated with both the liposome and the photosensitizer, entrapped ina liposome, complexed with a liposome, dispersed in a solutioncontaining a lipid, mixed with a lipid, combined with a lipid, containedas a suspension in a lipid, contained or complexed with a micelle, orotherwise associated with a lipid. The lipid or lipid/photosensitizerassociated compositions of the present invention are not limited to anyparticular structure in solution. For example, they may be present in abilayer structure, as micelles, or with a “collapsed” structure. Theymay also simply be interspersed in a solution, possibly formingaggregates which are not uniform in either size or shape.

Lipids are fatty substances which may be naturally occurring orsynthetic lipids. For example, lipids include the fatty droplets thatnaturally occur in the cytoplasm as well as the class of compounds whichare well known to those of skill in the art which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes. An example is the lipiddioleoylphosphatidylcholine.

Phospholipids may be used for preparing the liposomes according to thepresent invention and can carry a net positive charge, a net negativecharge or are neutral. Diacetyl phosphate can be employed to confer anegative charge on the liposomes, and stearylamine can be used to confera positive charge on the liposomes. The liposomes can be made of one ormore phospholipids.

In one embodiment, the lipid material is comprised of a neutrallycharged lipid. A neutrally charged lipid can comprise a lipid without acharge, a substantially uncharged lipid or a lipid mixture with equalnumber of positive and negative charges.

In one aspect, the lipid component of the composition comprises aneutral lipid. In another aspect, the lipid material consistsessentially of neutral lipids which is further defined as a lipidcomposition containing at least 70% of lipids without a charge. In otheraspects, the lipid material may contain at least 80% to 90% of lipidswithout a charge. In yet other preferred aspects, the lipid material maycomprise about 90%, 95%, 96%, 97%, 98%, 99% or 100% lipids without acharge.

In specific aspects, the neutral lipid comprises a phosphatidylcholine,a phosphatidylglycerol, or a phosphatidylethanolamine. In anotheraspect, the phosphatidylcholine comprises dioleoylphosphatidylcholine.

In other aspects the lipid component comprises a substantially unchargedlipid. A substantially uncharged lipid is described herein as a lipidcomposition that is substantially free of anionic and cationicphospholipids and cholesterol. In yet other aspects the lipid componentcomprises a mixture of lipids to provide a substantially unchargedlipid. Thus, the lipid mixture may comprise negatively and positivelycharged lipids.

Lipids suitable for use according to the present invention can beobtained from commercial sources. For example, dimyristylphosphatidylcholine (“DMPC”) can be obtained from Sigma Chemical Co.,dicetyl phosphate (“DCP”) is obtained from K & K Laboratories(Plainview, N.Y.); cholesterol (“Chol”) is obtained fromCalbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and otherlipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Preferably, chloroform is used as theonly solvent since it is more readily evaporated than methanol.

Phospholipids from natural sources, such as egg or soybeanphosphatidylcholine, brain phosphatidic acid, brain or plantphosphatidylinositol, heart cardiolipin and plant or bacterialphosphatidylethanolamine are preferably not used as the primaryphosphatide, i.e., constituting 50% or more of the total phosphatidecomposition, because of the instability and leakiness of the resultingliposomes.

“Liposome” is a generic term encompassing a variety of single andmultilamellar lipid vehicles formed by the generation of enclosed lipidbilayers or aggregates. Liposomes may be characterized as havingvesicular structures with a phospholipid bilayer membrane and an inneraqueous medium. Multilamellar liposomes have multiple lipid layersseparated by aqueous medium. They form spontaneously when phospholipidsare suspended in an excess of aqueous solution. The lipid componentsundergo self-rearrangement before the formation of closed structures andentrap water and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). However, the present invention also encompassescompositions that have different structures in solution than the normalvesicular structure. For example, the lipids may assume a micellarstructure or merely exist as nonuniform aggregates of lipid molecules.Also contemplated are lipofectamine-nucleic acid complexes.

Liposome-mediated oligonucleotide delivery and expression of foreign DNAin vitro has been very successful. Wong et al. (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells. Nicolau et al. (1987)accomplished successful liposome-mediated gene transfer in rats afterintravenous injection. Thus, it is contemplated that aliposome/photosensitive dye composition may be used to deliveradditional materials, including active, therapeutic or diagnostic agentsto a tissue. In one aspect the additional material is comprised in alipid construct, such as a liposome.

In certain embodiments of the invention, the lipid may be associatedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the lipid may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the lipid may be complexed or employed inconjunction with both HVJ and HMG-1. Such expression vectors have beensuccessfully employed in transfer and expression of an oligonucleotidein vitro and in vivo and thus are applicable for the present invention.Where a bacterial promoter is employed in the DNA construct, it alsowill be desirable to include within the liposome an appropriatebacterial polymerase.

Liposomes used according to the present invention can be made bydifferent methods. The size of the liposomes varies depending on themethod of synthesis. A liposome suspended in an aqueous solution isgenerally in the shape of a spherical vesicle, having one or moreconcentric layers of lipid bilayer molecules. Each layer consists of aparallel array of molecules represented by the formula XY, wherein X isa hydrophilic moiety and Y is a hydrophobic moiety. In aqueoussuspension, the concentric layers are arranged such that the hydrophilicmoieties tend to remain in contact with an aqueous phase and thehydrophobic regions tend to self-associate. For example, when aqueousphases are present both within and without the liposome, the lipidmolecules may form a bilayer, known as a lamella, of the arrangementXY—YX. Aggregates of lipids may form when the hydrophilic andhydrophobic parts of more than one lipid molecule become associated witheach other. The size and shape of these aggregates will depend upon manydifferent variables, such as the nature of the solvent and the presenceof other compounds in the solution.

Liposomes within the scope of the present invention can be prepared inaccordance with known laboratory techniques. Phospholipids (Avanti PolarLipids, Alabaster, Ala.), such as for example the preferred neutralphospholipid dioleoylphosphatidylcholine (DOPC), is dissolved intert-butanol. The lipid is then mixed with the photosensitizer,proteinaceous material, agent and/or other component(s). Tween 20 isadded to the lipid mixture such that Tween 20 is 5% of the composition'sweight. Excess tert-butanol is added to this mixture such that thevolume of tert-butanol is at least 95%. The mixture is vortexed, frozenin a dry ice/acetone bath and lyophilized overnight. The lyophilizedpreparation is stored at −20° C. and can be used up to three months.When required the lyophilized liposomes are reconstituted in 0.9%saline. The average diameter. of the particles obtained using Tween 20for encapsulating the lipid with the oligo is 0.7-1.0 μm in diameter.

Alternatively liposomes can be prepared by mixing liposomal lipids, in asolvent in a container, e.g., a glass, pear-shaped flask. The containershould have a volume ten-times greater than the volume of the expectedsuspension of liposomes. Using a rotary evaporator, the solvent isremoved at approximately 40° C. under negative pressure. The solventnormally is removed within about 5 min. to 2 hours, depending on thedesired volume of the liposomes. The composition can be dried further ina desiccator under vacuum. The dried lipids generally are discardedafter about 1 week because of a tendency to deteriorate with time.

Dried lipids can be hydrated at approximately 25-50 mM phospholipid insterile, pyrogen-free water by shaking until all the lipid film isresuspended. The aqueous liposomes can be then separated into aliquots,each placed in a vial, lyophilized and sealed under vacuum.

In other alternative methods, liposomes can be prepared in accordancewith other known laboratory procedures: the method of Bangham et al.(1965), the contents of which are incorporated herein by reference; themethod of Gregoriadis, as described in Drug Carriers in Biology andMedicine, G. Gregoriadis ed. (1979) pp. 287-341, the contents of whichare incorporated herein by reference; the method of Deamer and Uster(1983), the contents of which are incorporated by reference; and thereverse-phase evaporation method as described by Szoka andPapahadjopoulos (1978). The aforementioned methods differ in theirrespective abilities to entrap aqueous material and their respectiveaqueous space-to-lipid ratios.

The dried lipids or lyophilized liposomes prepared as described abovemay be dehydrated and reconstituted in a solution of inhibitory peptideand diluted to an appropriate concentration with an suitable solvent,e.g., DPBS. The mixture is then vigorously shaken in a vortex mixer.Unencapsulated additional materials, such as agents including but notlimited to hormones, drugs, nucleic acid constructs and the like, areremoved by centrifugation at 29,000×g and the liposomal pellets washed.The washed liposomes are resuspended at an appropriate totalphospholipid concentration, e.g., about 50-200 mM. The amount ofadditional material or active agent encapsulated can be determined inaccordance with standard methods. After determination of the amount ofadditional material or active agent encapsulated in the liposomepreparation, the liposomes may be diluted to appropriate concentrationsand stored at 4° C. until use. A pharmaceutical composition comprisingthe liposomes will usually include a sterile, pharmaceuticallyacceptable carrier or diluent, such as water or saline solution.

D. Formulations and Application

The present invention is generally directed to compositions comprisingat least one photosensitizer. The photosensitizer may be used without anadditional proteinacous, lipid, and/or active agent element, asendogenous lipids, proteins, polypeptides and peptides may provide areactable component upon photoactivation to form an adhesive tissue glueor biomatrix for delivery of additional components to a tissue. However,it is preferred that the composition comprise at least one proteinaceousand/or lipid material in combination with a photosensitizer. Althoughnot intended exclusively for use in tissue sealing, the compositions ofthe invention may be referred to as a tissue or surgical adhesive, glue,or sealant, or a wound sealant. All of the foregoing terms are usedherein to describe a combination of components capable of adhering,sealing, closing, apposing or otherwise joining, two or more soft tissueelements. A tissue glue thus preferably functions to promote, catalyzeor otherwise generally cause the formation of covalent bonds betweentissues, such as the edges of a wound or surgical incision, so that itpromotes the formation of a proteinaceous framework between tissueelements allowing the formation or reconstruction of a biological seal.

The compositions of the invention will generally comprise at least onebiocompatible lipid, protein, polypeptide, peptide and/or active agentcontaining composition in combination with an amount of at least onebiocompatible photosensitizer effective to promote the formation of anadhesive upon photoactivation. In other aspects, the combination willcomprise covalent and/or non-covalent conjugation of the lipid(s),protein(s), polypeptide(s), peptide(s) and/or active agent(s) with thephotosensitizer(s). In certain embodiments, the combination ofproteinaceous, active agent and/or lipid composition and at least onephotosensitizer is a mixture wherein the components are not oressentially not covalently bonded to each other prior tophotoactivation. In other embodiments, part or all of the proteinaceous,active agent and/or lipid composition is covalently bonded to the atleast one photosensitizer, or to each other in any combination. Incertain aspects, the conjugation between one or more proteinaceous,active agent and/or lipid components of the composition and the at leastone photosensitizer is a direct conjugation, such as at least onecovalent bond without additional linking moietie(s) connecting a lipid,active agent, protein, polypeptide or peptide to the photosensitizer, orto each other in any combination.

In other aspects, at least one additional atom or chemical groupcomprises at least one linking moiety that covalantly connects thelipid(s), active agent(s), protein(s), polypeptide(s) and/or peptide(s)to the photosensitizer, or to each other, in any combination. It ispreferred that such a linking moiety is short, i.e., that it separatesthe at least one lipid, active agent, protein, polypeptide, peptideand/or photosensitizer by about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 atomsin a chain, irrespective of any additional side chemical groups that maybe attached to the chain. However, longer linking moieties arecontemplated. In particular embodiments, one or more selected lipids,active agents, proteins, peptides and/or polypeptides may also beconjugated to each other in the proteinaceous and/or lipid composition.Chemical linking moieties and chemically bonding such a moiety toproteinaceous material and other components are known to those ofordinary skill in the art (see U.S. Pat. Nos. 5,880,270, 5,856,571,5,547,667, 5,387,578, 5,306,809 and 4,680,338, each incorporated hereinby reference). Linkers to lipids and other molecules are described inU.S. Pat. No. 5,840,674, incorporated herein by reference.

The novel compositions of the present invention may be formulated withany pharmacologically acceptable medium or diluent. Biocompatibleformulations of lipid, protein, polypeptide, peptide and/or active agentcontaining compositions and photosensitizers may variously includeaqueous solutions and physiological buffers and other agents, such asstabilizers and fatty acids. The protein content of the composition maybe adjusted according to the viscosity of the composition desired, andin view of the intended use of the composition and the nature of anytissues to which it may be applied. In general, photosensitizer, lipid,active agent, protein, polypeptide and/or peptide formulations of thecorrect viscosity are limited by the solubility of the proteinaceous,active agent, photosensitizer and/or lipid material. The solution is tobe thick enough to stay in the wound and not so thick as to clot in theneedle as it is being applied. The amount of each photosensitizer neededis proportional to the protein and/or lipid concentration and will bedetermined for each application. The proportion of protein tophotosensitizer is important, as too much photosensitizer will consumethe oxygen radicals before they have a chance to cause a reaction andinsufficient radicals will not generate enough reactive species tocomplete the reaction. The lifetime of the singlet oxygen will also varywith the concentration of the photosensitizer and of the solvent used.These variables can be determined for each application without undueexperimentation by those of skill in the art.

In certain embodiments, a preferred range of viscosity is from about 40to about 100 poise, with a range of about 80 to about 100 poise beingparticularly preferred. However, various viscosities are contemplated,including but not limited to, about 40, about 41, about 42, about 43,about 44, about 45, about 46, about 47, about 48, about 49, about 50,about 51, about 52, about 53, about 54, about 55, about 56, about 57,about 58, about 59, about 60, about 61, about 62, about 63, about 64,about 65, about 66, about 67, about 68, about 69, about 70, about 71,about 72, about 73, about 74, about 75, about 76, about 77, about 78,about 79, about 80, about 81, about 82, about 83, about 84, about 85,about 86, about 87, about 88, about 89, about 90, about 91, about 92,about 93, about 94, about 95, about 96, about 97, about 98, about 99,about 100 poise, and any range derivable therein.

In preparing a composition intended for application to a particularselected tissue, it is contemplated certain advantages may be gained byemploying a lipid, active agent, protein, polypeptide and/or peptideknown to be present within that selected tissue. One example of such acombination is the use of the crystallin lens protein in compositionsintended for application to the eye.

In certain aspects, ranges of composition that is applied includes, butis not limited to, of from about 10 mg glue composition/cm² to about 500mg /cm². A more preferred range would be of from about 20 mg/cm² toabout 100 mg/cm². In certain embodiments, the range of composition thatmay be applied includes, but is not limited to, about 1, about 2, about3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, about 20, about 21, about 22, about 23, about 24, about25, about 26, about 27, about 28, about 29, about 30, about 31, about32, about 33, about 34, about 35, about 36, about 37, about 38, about39, about 40, about 41, about 42, about 43, about 44, about 45, about46, about 47, about 48, about 49, about 50, about 51, about 52, about53, about 54, about 55, about 56, about 57, about 58, about 59, about60, about 61, about 62, about 63, about 64, about 65, about 66, about67, about 68, about 69, about 70, about 71, about 72, about 73, about74, about 75, about 76, about 77, about 78, about 79, about 80, about81, about 82, about 83, about 84, about 85, about 86, about 87, about88, about 89, about 90, about 91, about 92, about 93, about 94, about95, about 96, about 97, about 98, about 99, about 100, about 110, about120, about 130, about 140, about 150, about 160, about 170, about 180,about 190, about 200, about 210, about 220, about 230, about 240, about250, about 275, about 300, about 325, about 350, about 375, about 400,about 425, about 450, about 475, to about 500 mg/cm²or greater, and anyrange derivable therein.

The biocompatible proteinaceous, active agent, lipid and/orphotosensitizing components of the novel compositions disclosed hereinmay be in the form of a single component system such that they form asingle composition. Within the single composition, the components may bephysically separate or they may be physically combined, for example, bythe covalent attachment of a photosensitizer to a lipid, active agent,protein, polypeptide or peptide. This attachment may be achieved by anyone of a variety of chemical coupling means known to those of skill inthe art including, for example, the use of reducers, oxidants, acids,bases and other chemically reactive species such as HCO₃.

Alternatively, the compositions of the present invention may be in theform of double, triple or greater number component systems where bothindividual components are stored separately until use. In the lattercase, a single composition may still be formed by mixing, or combining,the lipid, protein, polypeptide, peptide and/or active agent containingand photosensitizing components some time prior to use. Alternatively,each component may be applied separately to the tissue area, or wound tobe joined, such that the active composition will only be formed onirradiation of the tissue itself, ie., it is only formed in situ. Itwill be understood that all such alternatives and combinations thereoffall within the scope of the present invention.

It is contemplated that the proteinaceous, active agent and/or lipidcomposition may comprise 1, 2, 3, 4, 5 or more selected lipids, activeagents, proteins, polypeptides and/or peptides. One or more of theselected lipids, active agents, proteins, polypeptides and/or peptidesmay be conjugated with the photosensitizers described herein, or thosethat are known to those of ordinary skill in the art. Thus, it iscontemplated that the compositions and methods disclosed herein may becombined, with, for example, additional photosensitizers and materialsincluding but not limited to those disclosed in U.S. Pat. Nos. 5,552,452and 5,913,884, each incorporated herein by reference.

Further aspects of the invention concerns kits for use in forming anadhesive connection between biological tissues or forming a biomatrix.Such kits will include a light-protected container comprising abiocompatible photosensitizer and a biocompatible lipid, protein,polypeptide, peptide and/or active agent containing composition asdescribed above. It is particularly important that the photosensitivecomponent be kept in the dark, i.e., in the light-protected container.Photosensitizers included in the kits in powdered form need to beprovided in water and water vapor tight containers and will be stored inrefrigerated environments, for example from about 4° C. to as cold asabout −80° C. However, as with the compositions, the kit may be in theform of a double, triple, or greater number component kit system wherethe components are stored separately until use. In this case, there isno requirement that the protein, polypeptide or peptide containingcomponent also be stored away from the light, and it may kept in aseparate container of any form. However, it is preferable that lipidsand light sensitive agents be stored away from the light.

Suitable containers are contemplated to include light-protected tubes;plastic, dark glass or otherwise light-protected bottles, or light-tightfoil wraps. Such containers may also comprise a means to apply theadhesive to the tissue, for example a collapsible tube, or a glasscontainer with a plunger device to dispense the adhesive comprising asyringe like device. It is contemplated that such a device may comprisea syringe in which the lipid, proteinaceous material, active agentand/or photosensitizer solutions are separated by a plate that containsserrations which will penetrate the photosensitizer containingcompartment and then allow mixing of the contents of that compartmentwith the contents of the protein containing compartment. In that theoperation of the kits will generally rely on photo-oxidative mechanismsand singlet oxygen generation, one may wish to store thephotosensitizer,the lipid, protein, polypeptide, peptide and/or activeagent containing compositions in a high oxygen atmosphere or in anatmosphere which has a higher oxygen concentration or tension thanusual. As with any biological formulation, it is advisable to storethese kits under refrigeration, such as at 4° C.

D. Tissue Welding

In still further embodiments, the present invention concerns methods forforming an adhesive connection between biological soft tissues orfilling in damage to other tissues. The invention may be used insurgical applications where precise adhesion is necessary and where theapplication of sutures or staples is inconvenient or less effective thantissue adhesive. The adhesive of the present invention has uses inclosing large wounds and tissue defects as in filling in a defect causedby debridement. Another use is as an artificial skin or covering agentto cover large, oozing surfaces inside or outside the body. Other usesinclude the repair of large, internal areas which are raw, or friableand which leak fluid and blood; reinforcement for a sutured anastomosis,thus rendering it water tight and bacteria tight; apposing parts of thebody which are normally held together by surface tension such as thelung and chest wall; and to seal leaking blebs in the lung which arevery difficult to treat.

All such methods and procedures can be described as “welding tissue”,“tissue welding”, a method to “weld tissue”, etc. In certainembodiments, the method involves application of the tissue glue to atleast one tissue. The “at least one tissue” may be a single tissue type,or a multiple tissues closely associated to form essentially onediscernable mass, layer, section or body of tissue to which the tissueglue may be applied. In other aspects, the “at least one tissue” may bea surface area, face or region of a tissue mass, layer, section or bodyof tissue. In certain aspects, the tissue welding process creates orpromotes adhesion of the at least one tissue to at least a secondtissue. The “at least a second tissue” may be a single tissue type, or amultiple tissues closely associated to form essentially one or morediscernable mass, layer, section or body of tissue that is separatedfrom the “at least one tissue.” In other aspects, the “at least a secondtissue” may be a surface area, face or region of a tissue mass, layer,section or body of tissue. Thus, it is possible that the tissue weldwill create or promote an adhesion between two or more ends, faces,surfaces areas, regions or the like of a single tissue mass or body oftissue. In certain aspects, tissue glue or one or more components of thetissue glue composition may be applied to either or both the at leastone tissue and the at least a second tissue. A non-limiting example ofsuch a tissue weld would be to connect two ends of an intestinal sheetof tissue to form a tube. Another non-limiting example would be totissue weld an incision or tear in the skin or muscle. Anothernon-limiting example, would be to weld three or more separated pieces oftissue together, wherein each of the tissue pieces is a different typeof tissue. Of course, one of ordinary skill in the art will recognizethat there are a multitude of possible combinations of tissue welds thatmay be made in light of the disclosures herein.

To perform such a tissue welding method or procedure, one would firstprepare a composition as described herein and then apply the compositionto the tissue(s) to be connected to form a tissue adhesive combination.One would then irradiate the tissue adhesive combination, i.e., applyelectromagnetic radiation to this area, in a manner effective to promotethe formation of an adhesive connection between the tissues. Theformation of an adhesive connection will be achieved by thephoto-oxidative effects of singlet oxygen generating proteinaceouscross-links between the amino acid components of the composition and/ortissue.

The present invention also encompasses methods for tissue closing orwound healing wherein the actual preparation of a separate lipid, activeagent, protein, polypeptide and/or peptide containing composition is notnecessary. Such methods utilize the lipids, polypeptides, peptidesand/or proteins located naturally within the tissue area as in situprotein containing compositions. To form an adhesive connection betweenbiological tissues in this manner one would form a biologicallyeffective amount of a tissue adhesive combination at the tissues byapplying only the photosensitizer and/or active agent component(s) tothe tissues. In certain aspects, ranges of glue composition that isapplied includes, but is not limited to, of from about 10 mg gluecomposition/cm ² to about 500 mg /cm². A more preferred range would beof from about 20 mg/cm² to about 100 mg/cm².

One would then again apply electromagnetic radiation the tissue adhesivecombination thus formed in a manner effective to promote the formationof an adhesive connection between the tissues.

In either of the methods described above, the amount of the compositionused will, naturally, be dependent on the tissue(s) to which it is beingapplied and on the size and nature of the wound or incision to be closedor the distance between the tissues to be apposed. The determination ofthe appropriate amount of the composition to be, applied will be knownto those of skill in the art in light of the present disclosure.Likewise, the application of the electromagnetic radiation will also beadapted to suit the particular circumstances of operation. It isgenerally envisioned that the time for performing a tissue closureprocedure in accordance herewith will be less than five minutes intotal. For example, the closure of an incision of about 5 mm in lengthis contemplated to require in the order of 1 to 2 minutes. The time ofirradiation of the composition will depend not only on the size of thelesion and the desired end strength of the bonds (i.e., greaterirradiation produces greater bond formation), but also on thecomposition itself, the characteristics of the wound, and thepractitioner's preference.

A particular advantage of the present invention is the precision withwhich it can be used. Other glues that have been used stick whereverthey are applied and much care must be taken to avoid the glue spillingover into areas where it is not desired. In the present invention, theglue is activated only where the practitioner applies the laser lightand this can be a very small area, i.e., on the order of about 2 mmdiameter. Thus any excess glue can be ignored or washed off. In this waythe area of actual tissue adhesion is precisely controlled and isdetermined by the steadiness of the application of the laser energy. Itis contemplated that even more precision is possible through theapplication of the laser energy by electronically controlled means. Theglue composition may also be applied in layers to gradually fill in adefect or gradually strengthen the wound. The electromagnetic radiationmay be delivered within the glue matrix through special delivery devicesor simply applied on top of the glue layer.

The electromagnetic radiation necessary to achieve photoactivation willgenerally have a wavelength from about 10 nm to about 810 nm and will bewithin the visual, infra red or ultra violet spectra. The radiation willbe supplied in the form of a monochromatic laser beam or other form ofelectromagnetic radiation source. The choice of energy source willgenerally be made in conjunction with the choice of photosensitizeremployed in the composition. For example, an excimer laser is suitablefor refractive surgeries. Suitable combinations of lasers andphotosensitizers will be known to those of skill in the art, and areexemplified, but not limited to, those shown in Table 2.

TABLE 2 Photosensitizer Laser Source Wavelength Any tetrapyrroleincluding but not limited Blue diode (460 nm) to: hematoporphyrin,chlorin, bacteriochlorin, phthalocyanine or naphthalocyanine Rose bengalor riboflavin Blue-Green Argon 488-514 nm Porphyrins or cyanines Greendiode (532 nm) Hematoporphyrin, protoporphyrin, other Red diode (630 nm)porphyrins, toluidine blue, malachite green, azure A, azure B, brilliantgreen, patent blue VF Chlorins, chlorin_(e)6, tetrahydroxyphenyl Reddiode 660 nm chlorin, methylene blue, Janus green, Benzoporphyrinderivative Red diode (690 nm) All of the dye listed under red diodesTunable dye laser (600-700 nm)

Tunable dye lasers are also used with the present invention. Theselasers can be tuned to emit generally any wavelength within a broadspectrum and allow for exact matching of dye and laser wavelength. Inother embodiments, the source of irradiation may be non-laser light(i.e., polychromatic light) from an incandescent, fluorescent or othersource which could be used to activate the glue/matrix composition. Inparticular embodiments the non-laser light source may be from a xenonbulb devices using filters and liquid light guides to deliver definedwavelength ranges depending on the filter. Application of thesetechnologies will be known to those of skill in the art.

The present invention is envisioned to be suitable for use in a varietyof surgical embodiments where one desires to seal, close, appose orotherwise join two or more portions of soft tissue, or to fill incavities. or damage to soft or hard tissues. The invention is consideredto be particularly suitable for use in microsurgery, such as, forexample, in surgical operations or maneuvers concerning the eye, smallvascular tissue, gastrointestinal tract, nerve sheaths, small ducts(e.g., urethra, ureter, bile ducts, thoracic duct) or even the innerear, teeth or gums. It can be used in areas where use of a suture in anon-sterile part of the body would help foster an infection such as inthe oral cavity. The invention may also be used in conjunction withordinary sutures to provide a better cosmetic closure, such as toprovide a smooth surface on top of the suture surface. It isparticularly suitable for procedures involving laparoscopic operationsor interventions such as laparoscopic (LP) cholecystectomy, LPnephrectomy, LP thoracic procedures, LP appendectomy, LP hernia repairs,LP tubal ligations and LP orbital surgeries. The present invention willalso be useful in retractive surgeries. However, these examples areclearly not limiting and the use of the methods and compositionsdescribed herein in connection with any type of wound closure orsurgical procedure is encompassed by the invention.

In yet further embodiments, the present invention concerns methods forcross-linking proteinaceous compounds, active agents and/or lipids. Suchmethods comprise forming an effective amount of a proteinaceous materialor lipid cross-linker combination at the proteins, polypeptides,peptides, active agents and/or lipids, to be cross-linked andirradiating the combination with electromagnetic radiation in a mannereffective to promote the formation of cross-links between the proteins,polypeptides, peptides, active agents and/or lipids. The at least oneproteinaceous compound, active agent and/or lipid cross-linkercombination may be formed by preparing a composition in accordanceherewith and applying the composition to the materials to becross-linked, or by applying only the photosensitizer component itselfto the proteins, polypeptides, peptides, active agents and/or lipids. Incertain embodiments, dimers, such as an albumin dimer may form duringcrosslinking. In further embodiments, albumin-collagen cross-linking mayoccur. It is also contemplated that in certain embodiments, type Iand/or type II photoprocesses may contribute to the process ofcrosslinking. The choice of electromagnetic radiation andphotosensitizer will be coordinated as discussed above and asexemplified by the combinations listed in Table 2.

Cross-linking proteins in this manner is contemplated to be of use in avariety of non-clinical embodiments including but not limited to, forexample, in cross-linking proteins for use in chromatographic columns orbeads, the immobilization of antibodies or antigens for diagnostic orpurification purposes, or for the fixing of proteins for microscopy.

E. Biomatrix Uses

It is contemplated that one or more photosensitizer, proteinaceousmaterial, lipid and/or active agent (e.g., cells, drugs, nucleic acidvectors, antigens, etc.) may be combined in the compositions and methodsof the present invention to form a biomatrix for delivery of one or moreactive agents to various tissue types. In certain embodiments, it iscontemplated that the active agents may comprise one or more therapeuticor diagnostic agents. Active agents include, but are not limited to, oneor more chemicals; drugs; proteins, polypeptides and peptides includingantibodies; hormones; nucleic acids including antisense oligonucleotidesand recombinant nucleic acid constructs; nutritional substances;antigens; and combinations thereof. These agents may be mixed with orcovalently bonded to either the proteinaceous component, lipid componentor dye component of the composition. The agent may be used alone andwithout the other non-dye components.

In certain embodiments, the agent is slowly released from thebiomatrix/tissue glue. The composition and light exposure may bemanipulated to allow optimum duration of the composition and thereforedrug delivery. Such adjustments of the consistancy and biodegradablenature of the matrix is within the ability of one of ordinary skill inthe art in light of the disclosures herein. In a particular embodiment,the biologic matrix may be loaded with a drug to enhance or retard woundhealing. The composition may be used to retard wound healing inoperation where scarring of an artificially created tract, shunt, and orfistua would not be desirable. The matrix may deliver a cytotoxic orcell growth inhibitory material, including but not limited to5-fluorouracil or mitomycin. Such cytotoxic drugs may be used in variousclinical applications, such as is used in glaucoma filtration surgery,or controlling the growth of skin keloids. Another use would be inproliferative vitreo-retinoapthy where the matrix may be applied toslowly release an agent such, including but not limited to vitamin E,that may inhibit or eliminate the proliferation of certain cells such asvitamin E (Larrosa J M. Et al., 1997). The matrix may also be used todeliver cortico steroids or non-steroidal anti-inflammatory agents, forapplications including but not limited to, control of post-operativeinflammation, such as in uveitic eyes after intra-ocular surgery orsurgery of the nervous system to control post-operative edema andinflammation. The composition may also be used to deliver antibiotics toa difficult to reach space, including but not limited to the vitreouscavity of the eye or to areas of chronic infection such asosteomyelitis. The matrix may also be used in drug or gene therapy todeliver long term depots of an agent over a long span of time to aspecific area/space. Particular agent that are contemplated includeanti-cancerous medications. In such uses the matrix would spare systemicexposure to the pharmaceutical agent and allow concentrated exposure fora relatively enhanced period of time. Again, this period of time may beadjusted based on composition of the matrix and level of lightactivation.

In one embodiment, the active agent is an antigen that is contacted witha host organism's immune system to promote immunity, or in some cases,tolerance to the antigen. Thus, a tissue glue or biomatrix vaccine iscontemplated.

In a preferred embodiment, the agent is a hormone involved in tissuegrowth or repair. Much progress has been made in identifying andcharacterizing the multitude of growth factors involved in the variousstages of the healing process. Recombinant DNA technology has led to theability to produce these polypeptides in a pure form without thenecessity of isolating them from human material. Consequently manyworkers have attempted to use these recombinant growth factors tostimulate processes of tissue repair and remedy deficiencies in woundhealing.

However it was quickly discovered that in order to be effective thesegrowth factors should remain in the tissue defect for a sufficientlylong time to allow the cellular process of healing to take place. Thistime should be at least about one hour, though longer periods such as 48hours and greater are preferable. However when growth factors aredirectly applied into tissue they remain only in position only for veryshort times, sometimes only minutes.

It may be that there are many cases where the actual tissue binding ofthe growth factor laser activated glue will be a distinct improvement,for instance allowing much more movement of the affected area withoutdislodging the glue. Examples of growth factors that may be used asagents in combination with the tissue glue include, but are not limitedto, transforming growth facto beta (isoforms 1, 2, or 3) (Foitzik etal., 1999; Yamamoto et al., 2000), basic fibroblast growth factor (Wang,1996), epidermal growth factor (Sheardown et al., 1997), vascularendothelial growth factor (Chawla et al., 1999), nerve growth factor(Mohammad et al., 2000), acidic fibroblast growth factor (Sellke et al.,1996), insulin like growth factor (Prisell et al., 1997), heparinbinding growth factors (Sakiyama-Elbert & Hubbell, 2000), brain-derivedneurotrophic factor and glial cell line-derived neurotrophic factor(Vejsada et al., 1998), platelet-derived growth factor (Khouri et al.,1993), leukemia inhibitory factor (Leong et al., 1999) and combinationsthereof. Examples of tissues that may be treated in this fashioninclude, but are not limited to, skin (Pierce et al., 1988), bone(Yamamoto et al., 2000), peripheral neurons and axons (Mohammad et al.,2000; Vejsada et al., 1998), central neurons and axons (Cheng et al.,1998), cartilage (Rayan & Hardingham, 1994), blood vessels (Chawla etal., 1999), cornea (Sheardown et al., 1997). Additional bioactivesubstances and methods of using a solder for delivery of them isdescribed in U.S. Pat. No. 5,713,891, incorporated herein by reference.

In other aspects, the agent is one or more living cells. The cells maybe shaped by the biomatrix into a monolayer or other conformation. It ispreferred that the biomatrix degrades in the tissue or organism it iscontacted with to allow the transplanted cells assume an optimum shapeor function in the tissue or organism. The biomatrix may be formed invitro or in vivo, and may serve as a reservoir of nutrients for livingcells, either for cells contained as an agent or for the tissue(s) towhich the biomatrix is applied. Thus, the biomatrix may serve as a typeof solid or semi-solid cell culture medium or therapeutic wounddressing. In certain embodiments, the biomatrix may be applied to woundsthat have traditionally healed poorly, including but not limited tocartilage, poorly vascularized areas, areas of lost tissue such as, forexample, skin lost due to burns. When applied to a tissue, it iscontemplated that the biomatrix can modulate the growth and morphologyof the transplanted cells, particularly when the biomatrix comprisesvarious growth factors, hormones, adhesive compositions that attract thetransplanted cells. In certain embodiments, the biomatrix may allow, oreven promote, the infiltration of host cells into the composition afterit is applied to a tissue.

The biomatrix may be varied in composition to be impermeable toadditional nutrient or aqueous media, or may allow such materials passinto or through the biomatrix. In certain embodiments, it is preferredthat the matrix is impermeable after photoactivation, such as use as atissue glue in organs including but not limited to the eye,gastrointestinal tract, blood vessels, etc.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 Photodynamic Tissue Adhesion with Chlorin_(e6) ProteinConjugates

This example demonstrates that the use of a chromophore, such as forexample, chlorin_(e6) (C_(e6)) may be effective in cross-linkingproteins by a photodynamic mechanism. Covalent conjugates between C_(e6)and proteins used as laser-activated solders form stronger tissue bondsthan noncovalent mixtures. The finding that addition of sodium azide tothe glue preparation reduces the leaking strength by more than 50% isattributed to quenching of singlet oxygen.

Preparation of Conjugates: C_(e6) was obtained from Porphyrin Products(Logan, Utah), N-hydroxy succinimide (NHS), dicyclohexylcarbodiimide,bovine fibrinogen, bovine serum albumin (BSA), and gelatin were fromSigma (St. Louis, Mo.). Frozen, nonpreserved, human cadaveric eyes wereobtained from the Illinois Eye Bank (Chicago). The reaction sequenceused to attach the C_(e6) molecules to the proteins covalently is shownin FIG. 1. All reactions were performed in the dark at room temperature.The NHS ester of C_(e6) was prepared by reacting 1.5 equivalent ofdicyclohexylcarbodiimide and 1.5 equivalents of NHS with 1 equivalent ofC_(e6) in dry dimethyl sulfoxide (DMSO) for 24 hr and was frozen inaliquots for further use. The concentration of the C _(e6)NHS in DMSOwas 100 mM. Proteins were dissolved in 0.1 M NaHCO₃ buffer (pH 9.3). ForBSA the concentration was 500 mg/ml, for fibrinogen 100 mg/ml, and forgelatin 200 mg/ml. A fivefold molar excess of C _(e6)NHS ester in DMSOwas added to the protein solution, which was allowed to stand overnight.The crude conjugate-solution was then dialyzed twice against 51phosphate-buffered saline (PBS) to remove unconjugated C_(e6) and DMSO.The conjugate between BSA and C_(e6) could be prepared easily anddialyzed to give a viscous dark green solution.

Mixtures of C_(e6) and proteins were prepared by dissolving C_(e6) in0.1 M NaOH to form a 100-mM solution, adding the requisite amount to theprotein solution in PBS and neutralizing with 0.1 M HCl. Conjugates werecharacterized by absorption spectroscopy after suitable dilution in PBS.

Conjugates, which contained approximately 6 mM BSA (400 mg/ml), had theappropriate consistency and viscosity for using as solders in incisions.The conjugate between gelatin and C_(e6) could not be prepared in a highenough concentration yield a sufficiently viscous solution. Theconjugate between fibrinogen and C_(e6) was substantially aggregated andunsuitable for use as solder. To explore the effect of these proteins onthe weld strength, gelatin was added to BSA−C_(e6), and fibrinogen wasmixed with BSA−C_(e6) and with free C_(e6). The compositions of theconjugates and mixtures that were used as solders are shown in Table 3.For Table 3, C_(e6) and protein concentrations were measured byabsorption spectroscopy after appropriate dilutions in 0.1 M NaOH and 1%sodium dodecyl sulfate using extinction coefficients for C_(e6) of150,000 at 400 nm, and for protein of 47,000 at 280 nm. Protein andC_(e6) data are in millimolar.

TABLE 3 DETAILS OF THE CONJUGATES AND MIXTURES USED AS SOLDERSProtein/C_(e6) Composition Protein C_(e6) Ratio BSA − C_(e6) conjugate5.2 6.2 0.84 BSA + C_(e6) mixture (1:1) 5.3 5.4 0.98 BSA + C_(e6)mixture (2.5:1) 5.9 2.3 2.56 BSA − C_(e6) conjugate + BSA 6.0 1.5 4 BSA− C_(e6) conjugate + gelatin 3.2 0.8 4 Fibrinogen + C_(e6) mixture 3.31.6 2.06 BSA − C_(e6) conjugate + 4.1 1.6 2.56 fibrinogen R-5-P +fibrinogen 0.9 5.7(r-5-P) 0.16(r-5-P)

Welding Procedure: To measure leaking pressures, an 18-gauge butterflyneedle was connected by plastic tubing to a water bottle. The innerpressure in the bottle was controlled by a hand-pumped sphygmomanometer.Nonpreserved cadaveric eyes were defrosted in room-temperature water.The butterfly needle was inserted in the vitreous cavity throughequatorial sclera, and the eye was pressurized to 25 to 30 mm Hg. Thecorneal epithelium was then removed at the wound site, and a caliper seton 5 mm used to make the extent of the incision on equatorial sclera,perpendicular to the limbus. All incisions were placed equidistant fromthe limbus, and areas of blue or thin sclera were avoided. Aperpendicular perforating incision was made with a 15° blade. Incisionswere made into the vitreous cavity and extended to the full length usingVannas scissors. The glue was applied in a thin layer to the surface ofthe wound with a tuberculin syringe and a 30-gauge needle. A smallamount was injected within the wound. Argon blue-green laser (488-514nm; Spectrum K3, HGM Medical Lasers, South Salt Lake City, Utah) at asetting of 0.6 W, 2-mm-diameter spot size, was applied to the wound for60 to 120 seconds in a continuous back and forth manner using a handheldfiberoptic probe. The exact power output of the fiber was measured usinga power meter (model 210; Coherent, Palo Alto, Calif.). Argon lasergoggles (Glendale Protective Technologies, Lakeland, Fla.) were worn,which allowed viewing of the fluorescence from the C_(e6) (emission 670nm). To set the remaining adhesive on the scleral surface, whichsurrounded the wound, additional laser was applied until loss offluorescence of the dye, which took another 30 to 45 seconds.

Leaking Pressure: Leaking pressures were then measured using thesphygmomanometer, which was increasingly pressurized in approximately10-mm Hg increments. Leaking pressure was recorded the moment the woundleaked air or fluid. This procedure was used in all eyes.

The results from the welding studies and determinations of the leakingpressures are shown in Table 4. The total energy delivered varied from24 to 57 J (60-120 seconds' exposure at powers ranging from 0.4 to 0.52W). At first, the adhesive strength of the welds produced by BSA−C_(e6)conjugate and BSA+C_(e6) mixture in which the molar ratios of protein toC_(e6) were roughly one to one were compared. However the leakingpressures obtained were low, and more BSA was added to the mixture ofBSA+C_(e6) to attain a molar ratio of 2.5:1 protein to C_(e6). Theleaking pressure showed a marked increase (Table 4). Theprotein-to-C_(e6) ratio was then modified to at least 2:1 protein toC_(e6) in the remaining preparations. These results were compared withthe preparation when (r-5-P) and fibrinogen were used at aprotein-to-r-5-P ratio of 0.16.

TABLE 4 DETAILS OF THE LEAKING STRENGTH AND APPLIED TOTAL ENERGY IN THEWELDING STUDIES Mean Leaking Pressure/ Total Mean Total Energy LeakingEnergy Composition Eyes Range Pressure Ratio BSA − C_(e6) conjugate 739-46 78.0 ± 11.9 1.75 ± 0.26 BSA + C_(e6) mixture (1:1) 3 24-46 63.0 ±24.3 1.77 ± 0.38 BSA − C_(e6) conjugate 7 24-46 127.1 ± 13.4 4.04 ± 0.52(2.5:1) BSA − C_(e6) 8 24-57 207.1 ± 11.1 5.59 ± 0.24 conjugate + BSABSA − C_(e6) conjugate + 11  26-53 101.8 ± 14.2 2.71 ± 0.47 gelatinFibrinogen + C_(e6) mixture 3 24-46 39.6 ± 12.5 1.1  ± 0.34 BSA − C_(e6)conjugate + 3 24-53 35.8 ± 1.8 0.93 ± 0.28 fibrinogen Fibrinogen + r-5-P6 24-46 139.5 ± 12.3 4.44 ± 0.53

Incisions in Table 4 were closed by welding using the solder compositionand total energies specified and leaking pressure measured with asphygmomanometer as described. Total energy is in joules, and leakingpressure is in millimeters of mercury.

The BSA+C_(e6) (2.5:1) was compared with the BSA−C_(e6) with added BSA,which raised the protein ratio to 4:1. Also investigated was a mixtureof BSA−C_(e6) and gelatin that had an overall protein-to-C_(e6)4:1. Theleaking pressure was measured as a function of applied total energy forthese three solder preparations, and the results are shown in FIG. 2.Although the green-blue argon laser is frequently used in ophthalmology,it can be readily seen from FIG. 2 that the wavelengths are suboptimalfor excitation of C_(e6). A total energy-dependent increase in leakingstrength was seen for all three solders, and the order of weld strengthwas found to be BSA−C_(e6)+BSA>BSA+C_(e6)>BSA−C_(e6)+gelatin. Alsoinvestigated as solders were mixtures of fibrinogen with C_(e6) (2:1)and with BSA−C_(e6) (2.5:1). These solders performed not as well asother mixtures, with a leaking pressure of only 30 mm Hg. These resultswere compared with the preparation consisting of fibrinogen and r-5-P ata ratio of 0.16 protein to r-5-P. The leaking pressure the inventorsobtained was similar to that reported (Khadem et al., 1994) (FIG. 3).These various mean leaking pressures were tested for significance with atwo-tailed unpaired Student's t-test. The strength of the weld fromBSA−C_(e6)+BSA was significantly greater than that from fibrinogen+r-5-P(P<0.05), BSA+C_(e6) (2.5:1; P<0.05), BSA−C_(e6) and gelatin (P<0.0005),whereas there was no significant difference between the strengths fromfibrinogen+r-5-P and from BSA+C_(e6) (2.5:1), whereas both weresignificantly stronger than BSA−C_(e6)+gelatin (P<0.05).

During the welding procedure there was a visually detectable change inthe appearance of the solder, which at first was dark green and becamebrownish green. Care had to be taken at the ends of the weld where themovement of the handheld fiberoptic probe naturally slowed down inpreparation for changing direction. This meant that the center of theincision received less light than the ends and that leakage of the woundstarted at this point. The consistency of the solder after welding wasfirm and smooth, and the tissue showed no thermal damage. This couldeasily be seen if the total energy or the power at which it wasdelivered was too great. The edges of the wound retracted and started towrinkle, and the weld failed, because the wound edges were no longer inapposition. Any solder resting on the cornea, that did not receive laserexposure continued to have a liquid, nonadhesive consistency.

EXAMPLE 2 Photodynamic Tissue Adhesion with Chlorin_(e6) ProteinConjugates

The adhesive strength of covalent conjugates to non-covalent mixturesbetween chlorin_(e6) and proteins in a laser activated soldering processare compared in this example.

The covalent and non-covalent mixtures of C_(e6) photosensitizercontained different molar ratios of albumin, gelatin and fibrinogen.These photodynamic glues were applied to 5 mm incisions along theequatorial sclera and activated with an Argon blue-green laser over atwo minutes period. Intra-ocular pressure was increased in 5 mmHgincrements until fluid or air escaped from the wound.

The albumin-chlorin_(e6) conjugate welds were significantly strongerthan non-covalent mixtures. The albumin−C_(e6) with added albuminprovided the highest bursting pressures (mean=207.1 mmHg) in comparisonto the other covalent conjugates (mean=88.8 mmHg) and non-covalentmixtures (mean=76.7 mmHg). The order of strength for the most effectivephotodynamic glues was determined to be:albumin−C_(e6)+albumin>albumin+C_(e6)>albumin−C_(e6)+gelatin−C_(e6).

These results demonstrate that covalent linkages between chlorin_(e6)photosensitizer and protein molecules formed stronger welds than thenon-covalent mixtures.

EXAMPLE 3 Tissue Glue as a Slow Release Biomatrix

In this example, a dye containing protein glue mixture containing aphamacologically active amount of the appropriate growth factor will beformulated. Experiments will be conducted in vitro to test the extent ofinactivation of the growth factor during the laser activation of theglue. It is expected that although some of the growth factor will beinactivated, enough will remain of the amount introduced to stimulatethe tissue repair or wound healing.

Preliminary experiments in the rat cornea show that the laser activatedglue remains in the incision for 48 hours, although much less than attime zero. This apparently fulfills the requirement of being a slowrelease vehicle with a defined rate of biodegradation.

EXAMPLE 4 Growth of Human Fetal RPE Patches on Different BiologicMatrices

The present example demonstrates that a biological matrix, such asformed by a tissue glue composition, may be used in celltransplantation. The present non-limiting example examines the growthpattern, orientation and ease of manipulation of transplanted humanfetal RPE (HFRPE) cells different biologic matrices.

Pieces of RPE monolayers, freshly isolated from human fetal eyes at17-23 weeks gestational age, were patched on the surface of the films(1.5-2.0 rabbit disc diameter) prepared from biodegradable photodynamicglue (J. Khadem et al.,1994) or autologous plasma clot. The cells werestudied by light and electron microscopy at different time periods afterpatching and compared to cultured cells from 2^(nd) passage.

Pieces of HFRPE demonstrated excellent initial attachment to the surfaceof the biologic glue film. At 48-72 hours new RPE cells appeared at theedges of the monolayer patch. The cells showed orderly growth in amonolayer pattern from high preservation of cellular shape andstructure. SEM showed polygonal cells with prominent microvilli on theapex. TEM revealed normal cellular structure with increasednuclear/cytoplasmic ratio. In contrast cultured cells showed lesspreservation of microvilli and appear elongated. Fresh HFRPE pieces andcultured HFRPE cells attached easily to, the surface of the plasma clotfilms, retaining their viability at 2 weeks, however no further growthwas noted. Both substrates possessed ease of manipulation utilizingvitreal forceps.

This example demonstrates that cells grown on the surface of thebiologic glue film from HRFPE patches show highly preserved cellularorientation and morphology and therefore may be useful intransplantation of RPE as a monolayer of primary cells. The consistenceof films prepared from the photodynamic biologic glue and plasma clotcan be easily handled and manipulated by the vitreal forceps. It iscontemplated that the specific tissue glue compositions disclosed hereinmay be used to grow and shape cells for transplantation, and shape thecells transplanted in tissues.

EXAMPLE 5 A new Model of Retinal Pigment Epithelium Transplantation withMicrospheres

In this example, a 3-dimensional culture system for human fetal retinalpigment epithelial (HFRPE) cells in the form of microspheres wasdeveloped, and cell growth in the subretinal space evaluated aftertransplantation. Fibrinogen, which is a powerful stimulator of cellattachment and proliferation (Gray et al., 1993), was used in thematrix. A multicellular spheroid was used, which is 1 form of a3-dimensional cell culture system (Hoffman, 1993).

Preparation of the matrix: Cross-linked fibrinogen films were preparedunder sterile conditions by mixing fibrinogen, 90 mg, and flavinmononucleotide, 1.3 mg (Sigma-Aldrich Corp. St. Louis, Mo.), in 5 mL ofdeionized water (Khadem et al., 1994). Four drops (−80 pl) of themixture were spread evenly on the bottom of a 30-cm Petri dish. Themixture was left under UV light for 12 hours. This allowed the formationof 20-to 50-g thick, yellowish, transparent, slightly sticky films thatcould easily be separated from the bottom of the dish with fine forceps.The film was cut into smaller 1×1-mm pieces that were used for HFRPEmonolayer implantation.

Separation and culture of HFRPE cells as microspheres: Human fetal eyesat 17 to 22 weeks of gestation were used in this study. The eyes wereenucleated and processed under aseptic conditions. They weresubsequently dissected circumferentially posterior to the ora serrata.After gentle removal of the vitreous and retina, RPE cells wereseparated from the choroid by forceps in large monolayer sheets. Nodigestive enzymes were used during the separation. After separation, thelarge HFRPE pieces were cut with microscissors into approximately 1×1-mmpieces. The HFRPE monolayer pieces were placed on the surface of thematrix and incubated in high-glucose Dulfecco modified eagle mediumsupplemented with 15% fetal bovine serum, levoglutamide, and acombination of penicillin G sodium and streptomycin sulfate. For thefirst 24 hours, the matrix films bearing the cells were kept attached tothe bottom of the culture dish. The films were then easily detached fromthe dish with forceps and kept in a floating state until microspheresformed. The films bearing the cells became rounded and formed oval orround conglomerates, i.e., microspheres, covered with HFRPE. Microsphereformation took place 7 to 10 days after attachment of HFRPE pieces tothe matrix. Three-week-old microspheres were used for transplantation.The growth pattern and morphologic characteristics of HFRPE microspheresattached to the floor of 8-well chamber slides (NUNC, Naperville, Ill.)were studied as in vitro controls.

Transplantation of HFRPE microspheres into the subretinal space: Allprocedures conformed to the Association for Research Guidelines inVision and Ophthalmology on the Use of Animals in Ophthalmic and VisionResearch as well as The University of Chicago guidelines for animalexperimentation. After a standard 3-port vitrectomy, a localizediatrogenic retinal bleb was created with balanced salt solution (He etal., 1993; el Dirini et al., 1992) in 1 eye of 9 albino and 9 pigmentedrabbits. A microsphere containing the HFRPE cells was introduced intothe vitreous cavity with, a blunt micropipette and transferred to thesubretinal space. Nine albino and 9 pigmented rabbits were used ascontrols. The controls underwent transplantation with a bare matrix ofcomparable size. Eyes that showed any signs of bleeding during theprocedure were excluded from the study. The eyes were evaluated at 7,14, and 30 days after surgery by indirect ophthalmoscopy and fundusphotograph. The rabbits underwent euthanasia, and the eyes wereenucleated at 7, 14 and 30 days after the surgery for histological andimmunohistochemical studies. Three albino and 3 pigmented rabbits wereeuthanatized at each point after the transplantation. Controls werefollowed up and euthanatized similarly. Paraffin-embedded and cryostatsections were used for these studies.

Immunohistochemistry: To identify the donor cells in the pigmented eyesthat underwent transplantation, the sections were stained withanti-human monoclonal HLA-ABC antibody specific for human tissue. Theantibody showed no cross-reaction with rabbit tissues. Monoclonalanti-human pancytokeratin was used as an epithelial marker. CD5monoclonal antibody used as an epithelial marker. CD5 monoclonalantibody (Sigma-Aldrich Corp), specific for rabbit panlymphocytes, wasused to assess the gross immune response.

For HLA-ABC (catalog number M; DAKO, Carpinteria, Calif.) and CD5immunostaining, the cryosections were fixed in cold acetone for 10minutes. Primary antibodies were used in a 1:10 dilution, and the slideswere incubated in a moist chamber at room temperature for 1 hour. Formonoclonal antipancytokeratin (catalog number C-2562; Sigma-AldrichCorp) immunostaining, the sections were stained in 10% bufferedformaldehyde solution for 10 minutes. The primary antibody was used at adilution of 1:20 for 2 hours in a moist chamber at 37° C. The secondaryantibodies used were sheep anti-mouse immunoglobulin-rhodamine B, 1:10,or immunoglobulin-fluorescein, 1:30 (Rosh Molecular Biochemical,Indianapolis, Ind.), for 1 hour at 37° C. After a final washing indistilled water, the specimens were covered with mounting medium andexamined under a microscope (model BH-2; Olympus, Osaka, Japan).Photographs were taken with a camera (model 35AD-4; Olympus, Japan).Film (Ektachromo 320T; Kodak, Rochester, N.Y.) was used for allfluorescence pictures. Monoclonal human anti-CD4 (Sigma-Aldrich Corp)was used as a negative control substrate. The in vitro control specimenswere stained similarly for anti-human monoclonal HLA-ABC antibody andfor monoclonal antipancytokeratin.

Results: Three albino and 3 pigmented rabbits (6 eyes) were studied ineach group that underwent transplantation and in each control group ateach period. In total, 36 eyes were studied.

Ophthalmoscopy: 0 to 7 Days: Polygonal human fetal retinal pigmentepithelial cells on the outer surface of the microsphere were seen at×200 magnification. At 7 days after the surgery, there was nohyperpigmentation around the microsphere. The transplanted microsphereappeared as a pigmented subretinal lesion. On day 7, subretinalhyperpigmentation was noted in 2 rabbit eyes in proximity to thetransplanted HFRPE microspheres. None of the eyes showed intraocularinflammation.

14 Days: At 14 days after the surgery, there is subretinalhyperpigmentation around the microsphere. Four of 6 eyes showedhyperpigmentation around the transplanted microsphere. In those eyes inwhich hyperpigmentation was present from day 7, an increase in its sizewith the formation of pseudopodia was noted around the donor tissuesource. No introcular inflammation was seen.

30 Days: At 1 month after surgery, no hyperpigmentation was noted aroundthe microsphere in this pigmented rabbit. Five of 6 eyes showedhyperpigmentation around the microsphere. No ophthalmoscopic evidence ofinflammation or infection was noted at the 30-day follow up.

In summary, the extent of hyperpigmentation around the microspheresvaried among the eyes, ranging from no hyperpigmentation (7 eyes; 3albino and 4 pigmented eyes) to prominent hyperpigmentation, with someextending as far as 3 to 4 disc diameters away from the initial donorsite.

A total of 11 of 18 rabbits that underwent transplantation showedsubretinal hyperpigmentation adjacent to the microspheres. In thecontrol eyes, no subretinal hyperpigmentation was noted. Starting fromday 7, prominent whitening due to a large disciform area ofchorioretinal atrophy, with no change in size with time, was seen in allcontrol pigmented rabbit eyes around the bare transplanted matrix.Similar chorioretinal atrophy was noted in the control albino eyes atthe site of the transplanted matrix.

Hisopathologic characteristics: There were no notable differences notedin the inflammatory response at various times after transplantation. Forbetter assessment of the sections, 3 tissue regions were defined: aregion over the microsphere, which included only the microsphere withoverlying retina and underlying choroid; a region close to themicrosphere, which included the area where the microsphere was alwaysseen with adjacent migrated cells; and a farther region, which includedsections where only donor cell monolayer was seen.

In the albino rabbit eye at 7 days after the transplantation, lightmicroscopy showed that the areas corresponding to the transplantedmicrospheres were composed of a circumscribed region of highly pigmentedHFRPE cells. The cells were residing in the subretinal space as thickmultilayers. Some of the cells have migrated into the retina. There islocal damage to the overlying retina.

An area in close proximity to the retinotomy site showed loss ofphotoreceptors. Two layers of pigmented cells were seen in thesubretinal space. At farther sites, the photoreceptors show betterpreservation. The pigmented cells formed 2 layers in the subretinalspace. Fundus photograph and corresponding light micrographs of freshfrozen sections from an albino rabbit eye 14 days after transplantationshowed areas of hyperpigmentation extending from the microsphere. Amonolayer of pigmented donor cells grew out from the microsphere in thesubretinal space was seen. Migrating cells are seen above the HFRPEmonolayer at the level of photoreceptors and appeared rounded. Residingand migrating cells were seen. The retina was artifactually detachedduring processing.

Loss of photoreceptors was typically noted immediately above and inclose proximity to the transplanted tissue. Migration of transplantedHFRPE cells into the overlying neurosensory retina was noted at the siteof microsphere implantation. Fragments of the matrix appeared aseosinophilic material between the HFRPE cells. Human fetal retinalpigment epithelial cells were seen in 2 eyes in close proximity to theinitial donor tissue at 7 days after the transplantation. At 14 daysafter the transplantation, the HFRPE cells were identified at fartherregions from the microsphere. In both albino and pigmented eyes, thedonor cells formed a monolayer in the subretinal space. Thiscorresponded to the hyperpigmentation site that was seenophthalmoscopically around the microsphere.

The cells forming the transplanted microspheres as well as pigmentedcells seen at the subretinal space showed positive immunostaining forHLA-ABC monoclonal antibodies. Migrating human fetal retinal pigmentepithelial cells that form a layer above the host retinal pigmentepithelium were seen. A negative control was used stained withirrelevant antibody.

Neurosensory retina was preserved above the pigmented cell monolayerslocated at the distant site from the microsphere.

The area with the transplanted microsphere was also studied by scanningelectron microscopy at 30 days after the transplantation in 2 albinoeyes. The retina was locally “glued” to the underlying microsphere, andthe microsphere appeared flattened. Two layers of RPE could beidentified. The cells in the top layer appeared more rounded, withfilamentous cell-cell-junctions, possibly representing donor tissue,while the bottom layer showed flatter, more polygonal cells, probablythe host RPE. The HFRPE cells that flew out from the microsphereappeared rounded and were of different sizes compared with the host RPEcells. They showed monolayer formation around the microsphere, and theyformed long filamentous cell-cell junctions. In some areas, themonolayers were not continuous; and in some areas, only 2 or 3 donorcells were seen.

The cellular response to the transplanted tissue was strictly local andwas present around the microsphere, mainly in the underlying choroid.Compared with the controls, there was minimal choroidal thickening withmononuclear cell infiltration beneath the microsphere itself. Noinflammatory or lymphoctytic responses were seen in the areas where theHFRPE cells were distributed as monolayers.

A striking difference was noted in the control eyes in which only barematrix microspheres were transplanted. The subretinal human fetalretinal pigment epithelial microsphere in an albino rabbit eye showedthe donor cells that migrated from the initial source. Choroidalinfiltration appears less compared with the control. The controlpigmented rabbit eye transplanted with bare microsphere matrix had thewhole area infiltrated with inflammatory cells.

A markedly thickened, infiltrated choroid was evident under thetransplanted matrix, with loss of photoreceptors in the overlyingretina. Lymphocytes and other mononuclear cells invaded the entire areaof the subretinal space and the retina around the matrix. This couldrepresent a mixed nonspecific inflammatory and immune response. Thiscellular response was local and was confined to the area of thetransplanted bare matrix. The reaction was more severe than in the eyesthat underwent HFRPE transplantation. Multiple cells with engulfedeosinophilic material, possibly the matrix, were identified in the area.At 30 days after the surgery, some of the control eyes showedchorioretinal atrophy, with no extracellular matrix present.Immunostaining with CD5 monoclonal antibody, which recognizes rabbitpanlymphocytes, showed more intense lymphocytic infiltration in controleyes than in the eyes that underwent HFRPE transplantation. Fluorescenceimage from an eye that underwent transplantation shows only minimalstaining against CD5 compared with the control eye transplanted withmatrix only. A negative control was stained with irrelevant antibody.

Subretinal RPE transplantation has shown promise in the rescue ofoverlying receptors in some experimental degenerative retinal diseases(Tamai, 1996). This may be important in the management of variousdiseases affecting the RPE. A 3-dimensional culture system offers a newapproach for the provision of donor tissue into the subretinal space.

The transfer of the HFRPE cell-containing microspheres into thesubretinal space is simple and reproducible. The adjustable size andspherical shape of the donor tissue makes it easy to insert into thesubretinal space. Because the HFRPE cells in the model form compacttissue, conglomerates, there is less chance for cell reflux(Wongpichedchai et al., 1992). Other studies (Gouras et al., 1992) ofRPE transplantation, there was no evidence of donor cell proliferationor migration in the subretinal space. Some studies indicate that thesubretinal space is an immune privileged environment where cellproliferation is kept under strict control. Transplanted cells may needsome kind of initial stimulation to migrate or proliferate actively inthis environment. Fibrinogen and the 3-dimensional state of HFRPE tissuecould contribute to the ability of the cells using the techniques inthis example to grow out from the initial source.

Studies done in the laboratory provide indirect evidence of theimportance of the modulatory effect of the matrix on cell behavior inthe subretinal space. Human fetal retinal pigment epithelial cells grownas microspheres on a synthetic polymer matrix showed notably lesspotential for subretinal spread compared with cells grown on afibrinogen matrix (Williamson et al., 1998). Some cell types reexpresstheir original in vivo characteristics in a 3-dimensional culture andmaintain cell-specific functions that are lost in monolayer cultures.The cells in 3-dimensional cultures express increased DNA synthesis andproliferation (Mered et al., 1980; Tamura et al., 1995). Donor cellsprovided to the recipient as 3-dimensional culture systems showprolonged survival and the ability to migrate from the initial sourceand establish themselves among the host tissues (Wintermantel et al.,1992; Fawcett et al., 1995). In addition, fibrinogen is a known potentstimulator for cell proliferation and migration (Sporn et al., 1995).Related to it, fibronectin is an important constituent of the Bruchmembrane and the surrounding RPE (Campochiaro et al., 1986). Human fetalretinal pigment epithelial cells grown on cross-linked fibrinogen matrixin a 3-dimensional state with tight cell-cell contacts may; becomeactivated (Koller and Papoutsakis, 1995; Olive and Durand, 1994), andpossess the potential for migration and proliferation after beingbrought into the subretinal space. Although there was notable damage tothe overlying retina, it appeared to be only local and restricted to thesite of the microsphere. The damage can be comparable in size with alarge laser bum. The retina appears preserved, however, above the HFRPEmonolayers at more distant sites from the microsphere. The growth ofHFRPE cells outside the maternal source of donor tissue, shown in thesestudies, provide an opportunity to transplant microspheres in anextrafoveal region, with secondary spreading to the subfoveal space.

The ophthalmoscopic observation of subretinal hyperpigmentation aroundthe transplanted microspheres corresponded histologically to a monolayerof pigmented cells in albino rabbits. Immunohistochemically, thetransplanted cells were identified by staining for HL-k-ABC antibody,and similarly showed migrating HFRPE cells from the initial source withmonolayer formation close to the transplanted microsphere.

All control eyes transplanted with bare matrix showed a notably higherinflammatory response and increased lymphocytic infiltration comparedwith the HFRPE transplanted eyes. Retinal pigment epithelium canmodulate the functions and behavior of other cells, such as lymphocytes,vascular endothelial cells, and macrophages (Liversidge et al., 1994;Liversidge et al., 1993; Sakamoto et al., 1995). Recent studies (Ochalekand von Kleist, 1994) have shown that some tumor cells grown asspheroids show increased resistance to lymphocyte lysis and inhibitionof lymphocyte penetration compared with the cells grown as monolayers.Human fetal retinal pigment epithelial cells transplanted asmulticellular spheroids, i.e. microspheres, may similarly possesslymphocyte inhibitory qualities. Retinal pigment epithelium cells havebeen shown to release transforming growth factor β family proteins(Anderson et al., 1995) that have immunosuppressive functions and thatcan inhibit neovascularization (Yoshimura et al., 1995; Seaton et al.,1994). In addition, cell types grown in a w-dimensional culture systemshow increased leves of intracellular cytokines, including transforminggrowth factors. Human fetal retinal pigment epithelial cells cover thematrix, possibly preventing its direct contact with the subretinaltissues, resulting in less intense inflammation compared with controls.Some studies (Tamai, 1996) explain he rescuing effect of RPEtransplantation to cytokine release by healthy donor cells.Three-dimensional cultures of HFRPE cells may be a better source of thecumulative release of different trophic cytokines (Ness et al., 1994),compared with monolayers, due to the high accumulation of healthy cells;

In conclusion, the provision of donor cells into the subretinal space asmicrospheres is reproducible and technically easy, and it may decreasethe chances for iatrogenic damage to the retina. The donor cells canspread and survive in the subretinal space for at least 1 month.

All of the compositions and/or methods and/or apparatus disclosed andclaimed herein can be made and executed without undue experimentation inlight of the present disclosure. While the compositions and methods ofthis invention have been described in terms of preferred embodiments, itwill be apparent to those of skill in the art that variations may beapplied to the compositions and/or methods and/or apparatus and in thesteps or in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

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What is claimed is:
 1. A method to weld tissue together, comprising thesteps of: (a) applying to at least one tissue a composition comprisingat least one photosensitizer and at least one proteinaceous compound orat least one lipid; and (b) irradiating said composition withelectromagnetic energy wherein said irradiating promotes adhesion ofsaid tissue to at least a second tissue and wherein; (i) the ratio oftotal proteinaceous molecules in said composition and said at least onephotosensitizer is from about 10:1 to about 1:10; (ii) the compositionis applied at of from about 10 mg said composition per cm² of saidtissue to about 500 mg said composition per cm² of said tissue; or (iii)said composition has a viscosity of about 40 to about 100 poise beforesaid irradiation.
 2. The method of claim 1, wherein said photosensitizeris a cationic azine mon-azo dye or derivative thereof.
 3. The method ofclaim 2, wherein said cationic azine mono-azo dye is neutral red.
 4. Themethod of claim 2, wherein said cationic azine mono-azo dye is JanusGreen.
 5. The method of claim 1, wherein said photosensitizer is atri-arylmethane-dye or derivative thereof.
 6. The method of claim 5,wherein said tri-arylmethane dye is Malachite Green, Brilliant Green,Crystal Violet, basic fuschin, pararosaniline acetate, methyl green ornew fuschin.
 7. The method of claim
 5. wherein said tri-arylmethane dyeis Malachite Green.
 8. The method of claim 5, wherein saidtri-arylmethane dye is a zwitterionic triarylmethane dye.
 9. The methodof claim 8, wherein said zwitterionic triarylmethane dye is patent blueVF.
 10. The method of claim 1, wherein said photosensitizer is atetrapyrrole or a derivative thereof.
 11. The method of claim 10,wherein said tetrapyrrole is a porphyrin, chlorin, bacteriochlorin,phthalocyanine, naphthalocyanine, texaphyrin, verdin, purpurin orpheophorbide.
 12. The method of claim 11, wherein said tetrapyrrole is achlorin.
 13. The method of claim 12, wherein said tetrapyrrole ischlorin_(e)6.
 14. The method of claim 1, wherein said photosensitizer isa cationic thiazine dye or derivative thereof.
 15. The method of claim14, wherein said cationic thiazine dye is Azure A, Azure B, Azure C,Brilliant Green, Crystal Violet or Patent Blue VF.
 16. The method ofclaim 1, wherein said composition further comprises at least a secondphotosensitizer.
 17. The method of claim 16, wherein said at least asecond photosensitizer is a cationic azine mon-azo dye, atri-arylmethane dye, a tetrapyrrole, a cationic thiazine dye, xanthine,an anthracenedione, an anthrapyrazole; an aminoanthraquinone, aphenoxazine dye, a phenothiazine derivative, a chalcogenapyrylium dye orderivatives thereof.
 18. The method of claim 1, wherein said compositioncomprises at least one proteinaceous compound.
 19. The method of claim18, wherein said proteinaceous compound comprises at least one peptide,polypeptide or protein.
 20. The method of claim 19, wherein saidproteinaceous compound comprises at least one protein.
 21. The method ofclaim 20, wherein said protein is albumin, fibrinogen or gelatin. 22.The method of claim 21, wherein said protein is albumin.
 23. The methodof claim 1, wherein said composition is a non-covalent mixture.
 24. Themethod of claim 1, wherein at least one covalent bond conjugates saidphotosensitizer to said proteinaceous composition or said lipid.
 25. Themethod of claim 24, wherein said covalent bond is part of a linkingmoeity.
 26. The method of claim 24, wherein said composition comprisesat least a second proteinaceous compound not covalently conjugated tosaid photosensitizer.
 27. The method of claim 26, wherein theproteinaceous compound covalently conjugated to said photosensitizer isthe same type as the proteinaceous compound not covalently conjugated tosaid photosensitizer.
 28. The method of claim 1, wherein the ratio oftotal proteinaceous molecules in said composition and said at least onephotosensitizer is from about 10:1 to about 1:10.
 29. The method ofclaim 28, wherein the ratio of total proteinaceous molecules in saidcomposition and said at least one photosensitizer is from about 3:1 toabout 1:1.
 30. The method of claim 29, wherein the ratio of totalproteinaceous molecules in said composition and said at least onephotosensitizer is about 2:1.
 31. The method of claim 1, wherein saidcomposition comprises at least one lipid.
 32. The method of claim 31,wherein said lipid further comprises at least one proteinaceouscompound.
 33. The method of claim 32, wherein said proteinaceouscompound is a lipoprotein.
 34. The method of claim 1, wherein saidcomposition further comprises at least one therapeutic agent.
 35. Themethod of claim 34, wherein said agent is a chemical, a drug, aproteinaceous molecule, a nucleic acid, a lipid, an antibody, anantigen, a hormone, a nutritional substance, a cell or a combinationthereof.
 36. The method of claim 35, wherein said agent is a hormone.37. The method of claim 36, wherein said hormone is a growth factor. 38.The method of claim 37, wherein said growth factor is transforminggrowth factor beta, basic fibroblast growth factor, epidermal growthfactor, vascular endothelial growth factor, nerve growth factor, acidicfibroblast growth factor, insulin like growth factor, heparin bindinggrowth factor, brain-derived neurotrophic factor, glial cellline-derived neurotrophic factor, platelet-derived growth factor,leukemia inhibitory factor or combination thereof.
 39. The method ofclaim 35, wherein said agent is a cell.
 40. The method of claim 39,wherein said cell is an embryonic cell.
 41. The method of claim 1,wherein said tissue is skin, bone, neuron, axon, cartilage, blood vesselor cornea.
 42. The method of claim 1, wherein said second tissue is thesame tissue type as said at least a first tissue.
 43. The method ofclaim 1, wherein said second tissue is a different tissue type as saidat least one tissue.
 44. The method of claim 1, wherein said compositionhas a viscosity of about 40 to about 100 poise before said irradiation.45. The method of claim 1, wherein said composition is applied at offrom about 10 mg said composition per cm² of said tissue to about 500 mgsaid composition per cm² of said tissue.
 46. The method of claim 45,wherein said composition is applied of from about 20 mg said compositionper cm² of said tissue to about 100 mg said composition per cm² of saidtissue.